Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.