STUDY QUESTION: Which processes and transcription factors specify the first and second lineage segregation events during human preimplantation development? SUMMARY ANSWER: Differentiation into trophectoderm (TE) cells can be initiated independently of polarity; moreover, TEAD1 and YAP1 co-localize in (precursor) TE and primitive endoderm (PrE) cells, suggesting a role in both the first and the second lineage segregation events. WHAT IS KNOWN ALREADY: We know that polarity, YAP1/GATA3 signalling and phospholipase C signalling play a key role in TE initiation in compacted human embryos, however, little is known about the TEAD family of transcription factors that become activated by YAP1 and, especially, whether they play a role during epiblast (EPI) and PrE formation. In mouse embryos, polarized outer cells show nuclear TEAD4/YAP1 activity that upregulates Cdx2 and Gata3 expression while inner cells exclude YAP1 which upregulates Sox2 expression. The second lineage segregation event in mouse embryos is orchestrated by FGF4/FGFR2 signalling which could not be confirmed in human embryos; TEAD1/YAP1 signalling also plays a role during the establishment of mouse EPI cells. STUDY DESIGN, SIZE, DURATION: Based on morphology, we set up a development timeline of 188 human preimplantation embryos between Day 4 and 6 post-fertilization (dpf). The compaction process was divided into three subgroups: embryos at the start (C0), during (C1), and at the end (C2) of, compaction. Inner cells were identified as cells that were entirely separated from the perivitelline space and enclosed by cellular contacts on all sides. The blastulation process was divided into six subgroups, starting with early blastocysts with sickle-cell shaped outer cells (B0) and further on, blastocysts with a cavity (B1). Full blastocysts (B2) showed a visible ICM and outer cells referred to as TE. Further expanded blastocysts (B3) had accumulated fluid and started to expand due to TE cell proliferation and zona pellucida (ZP) thinning. The blastocysts then significantly expanded further (B4) and started to hatch out of the ZP (B5) until they were fully hatched (B6). PARTICIPANTS/MATERIALS, SETTING, METHODS: After informed consent and the expiration of the 5-year cryopreservation duration, 188 vitrified high quality eight-cell stage human embryos (3 dpf) were warmed and cultured until the required stages were reached. We also cultured 14 embryos that were created for research until the four- and eight-cell stage. The embryos were scored according to their developmental stage (C0-B6) displaying morphological key differences, rather than defining them according to their chronological age. They were fixed and immunostained for different combinations of cytoskeleton (F-actin), polarization (p-ERM), TE (GATA3), EPI (NANOG), PrE (GATA4 and SOX17), and members of the Hippo signalling pathway (YAP1, TEAD1 and TEAD4). We choose these markers based on previous observations in mouse embryos and single cell RNA-sequencing data of human embryos. After confocal imaging (LSM800, Zeiss), we analysed cell numbers within each lineage, different co-localization patterns and nuclear enrichment. MAIN RESULTS AND THE ROLE OF CHANCE: We found that in human preimplantation embryos compaction is a heterogeneous process that takes place between the eight-cell to the 16-cell stages. Inner and outer cells are established at the end of the compaction process (C2) when the embryos contain up to six inner cells. Full apical p-ERM polarity is present in all outer cells of compacted C2 embryos. Co-localization of p-ERM and F-actin increases steadily from 42.2% to 100% of the outer cells, between C2 and B1 stages, while p-ERM polarizes before F-actin (P < 0.00001). Next, we sought to determine which factors specify the first lineage segregation event. We found that 19.5% of the nuclei stain positive for YAP1 at the start of compaction (C0) which increases to 56.1% during compaction (C1). At the C2 stage, 84.6% of polarized outer cells display high levels of nuclear YAP1 while it is absent in 75% of non-polarized inner cells. In general, throughout the B0-B3 blastocyst stages, polarized outer/TE cells are mainly positive for YAP1 and non-polarized inner/ICM cells are negative for YAP1. From the C1 stage onwards, before polarity is established, the TE marker GATA3 is detectable in YAP1 positive cells (11.6%), indicating that differentiation into TE cells can be initiated independently of polarity. Co-localization of YAP1 and GATA3 increases steadily in outer/TE cells (21.8% in C2 up to 97.3% in B3). Transcription factor TEAD4 is ubiquitously present throughout preimplantation development from the compacted stage onwards (C2-B6). TEAD1 displays a distinct pattern that coincides with YAP1/GATA3 co-localization in the outer cells. Most outer/TE cells throughout the B0-B3 blastocyst stages are positive for TEAD1 and YAP1. However, TEAD1 proteins are also detected in most nuclei of the inner/ICM cells of the blastocysts from cavitation onwards, but at visibly lower levels as compared to that in TE cells. In the ICM of B3 blastocysts, we found one main population of cells with NANOG+/SOX17-/GATA4- nuclei (89.1%), but exceptionally we found NANOG+/SOX17+/GATA4+ cells (0.8%). In seven out of nine B3 blastocysts, nuclear NANOG was found in all the ICM cells, supporting the previously reported hypothesis that PrE cells arise from EPI cells. Finally, to determine which factors specify the second lineage segregation event, we co-stained for TEAD1, YAP1, and GATA4. We identified two main ICM cell populations in B4-6 blastocysts: the EPI (negative for the three markers, 46.5%) and the PrE (positive for the three markers, 28.1%) cells. We conclude that TEAD1 and YAP1 co-localise in (precursor) TE and PrE cells, indicating that TEAD1/YAP1 signalling plays a role in the first and the second lineage segregation events. LIMITATIONS, REASONS FOR CAUTION: In this descriptive study, we did not perform functional studies to investigate the role of TEAD1/YAP1 signalling during the first and second lineage segregation events. WIDER IMPLICATIONS OF THE FINDINGS: Our detailed roadmap on polarization, compaction, position and lineage segregation events during human preimplantation development paves the way for further functional studies. Understanding the gene regulatory networks and signalling pathways involved in early embryogenesis could ultimately provide insights into why embryonic development is sometimes impaired and facilitate the establishment of guidelines for good practice in the IVF lab. STUDY FUNDING/COMPETING INTERESTS: This work was financially supported by Wetenschappelijk Fonds Willy Gepts (WFWG) of the University Hospital UZ Brussel (WFWG142) and the Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO, G034514N). M.R. is doctoral fellow at the FWO. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.