Human embryonic stem cells (hESCs) serve as an ideal cell source for generating hematopoietic stem cells (HSCs). In embryonic hematopoiesis, hemogenic endothelium has been identified as a source of HSCs, yet the regulatory mechanisms remain elusive. Here, through dynamic gene expression profiling analysis and verification, we find that ELTD1 expression parallels genes related to the specification of hemogenic endothelium progenitors (HEPs) from hESCs and is highly expressed in the HEPs. We then investigate the impact of ELTD1 on the hematopoietic differentiation of hESCs via gain- and loss-of-function experiments. Knockdown or deletion of ELTD1 mediates hESC hematopoiesis by specifically facilitating the generation of HEPs, thus promoting endothelial-to-hematopoietic transition to generate more hematopoietic cells. Besides, the overexpression of ELTD1 serves to further solidify this conclusion. Mechanistically, we demonstrate that ELTD1 exerts its function through the Wnt signaling pathway by bioinformatic analyses and functional studies. In addition, our results demonstrate a protein-protein interaction between ELTD1 and HPIP and further reveal that HPIP modulates the Wnt signaling pathway through LEF1. Collectively, these findings indicate that the ELTD1-HPIP-LEF1-Wnt regulatory axis acts as a novel mechanism regulating HEP generation during early hematopoietic differentiation of hESCs, providing new insights into the molecular mechanisms underlying human hematopoiesis.