During early development, the first hematopoietic stem cells (HSCs) emerge and derive from hemogenic endothelial (HE) cells, through a process called endothelial to hematopoietic transition (EHT). Adult HSC quiescence, maintenance and differentiation are tightly regulated by changes in metabolism. However, the role of metabolic cues during the first emergence of HSCs from HE cells is still unclear. Using human iPS-derived HE cells, we showed that during EHT, there is a gradual and simultaneous increase in glycolysis and oxidative phosphorylation. Interestingly, blocking glycolysis or glutaminolysis severely impaired hematopoietic differentiation. While the rescue of glutamine deprivation by α-ketoglutarate boosted myeloid/lymphoid cell formation, we found that a combination of α-ketoglutarate and nucleotides was required for erythropoietic commitment of HE cells. Both in vitro and in vivo, differentially modulating pyruvate catabolism directed HE cell fate toward either erythroid or myeloid/lymphoid outcomes. Thus, our findings uncover that metabolism plays a crucial role during human EHT and nutrients modulate lineage specification during hematopoietic emergence.