Little is known about hematopoietic stem cell (HSC) development from mesoderm. To gain more information on the intraembryonic HSC site of origin, we purified multipotent hematopoietic progenitors from the aorta-gonads-mesonephros (AGM) of mice. This population, expressing c-Kit, AA4.1, CD31, and CD41, but not Flk1, and mainly negative for CD45, proved capable of long-term reconstitution in sublethally irradiated Rag2gammac(-/-) recipients. We assigned the expression of GATA-2, GATA-3, and lmo2 to AGM-HSC, whereas erythromyeloid progenitors express only GATA-2. This unique combination of surface markers and transcription factors could be allocated in the AGM to the intraaortic clusters and the subaortic patches underlying aortic endothelial cells. Taken together, those data indicate that embryonic HSCs (i) differ from their fetal liver and adult counterpart by the low expression of CD45, (ii) do not colocalize with aortic endothelial cells as previously thought, and (iii) are localized, at 10.5 days postcoitum, in the splanchnic mesoderm underlying aortic endothelial cells, within GATA-3(+)CD31(+) cell clusters.