Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1−/− mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1−/− cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed. Display omitted •Hemogenic endothelium (HE) and pro-HSCs emerge in Ncx1−/− embryos lacking circulation•Ncx1−/− pro-HSCs are transcriptionally and functionally impaired•The HE to pro-HSC transition involves a glycolysis to OxPhos metabolic shift•Activation of glycolysis reduces hematopoietic output of E9.5 intraembryonic precursors Azzoni et al. report that hematopoietic stem cell precursors (pro-HSC) still emerge in the absence of circulation, but are functionally impaired and do not activate the hematopoietic program. Single-cell transcriptomics indicate that the onset of circulation induces a metabolic switch that is required for hematopoietic development.