To detect as yet unidentified cell-surface molecules specific to hematopoietic stem cells (HSCs), a modified signal sequence trap was successfully applied to mouse bone marrow (BM) CD34 − c-Kit + Sca-1 + Lin − (CD34 − KSL) HSCs. One of the identified molecules, Endomucin, is an endothelial sialomucin closely related to CD34. High-level expression of Endomucin was confined to the BM KSL HSCs and progenitor cells, and, importantly, long-term repopulating (LTR)–HSCs were exclusively present in the Endomucin + CD34 − KSL population. Notably, in the yolk sac, Endomucin expression separated multipotential hematopoietic cells from committed erythroid progenitors in the cell fraction positive for CD41, an early embryonic hematopoietic marker. Furthermore, developing HSCs in the intraembryonic aorta-gonad-mesonephros (AGM) region were highly enriched in the CD45 − CD41 + Endomucin + fraction at day 10.5 of gestation (E10.5) and in the CD45 + CD41 + Endomucin + fraction at E11.5. Detailed analyses of these fractions uncovered drastic changes in their BM repopulating capacities as well as in vitro cytokine responsiveness within this narrow time frame. Our findings establish Endomucin as a novel cell-surface marker for LTR-HSCs throughout development and provide a powerful tool in understanding HSC ontogeny.