Surface antigens on hematopoietic stem cells (HSCs) enable prospective isolation and characterization. Here, we compare the cell-surface phenotype of hematopoietic repopulating cells from murine yolk sac, aorta-gonad-mesonephros, placenta, fetal liver, and bone marrow with that of HSCs derived from the in vitro differentiation of murine embryonic stem cells (ESC-HSCs). Whereas c-Kit marks all HSC populations, CD41, CD45, CD34, and CD150 were developmentally regulated: the earliest embryonic HSCs express CD41 and CD34 and lack CD45 and CD150, whereas more mature HSCs lack CD41 and CD34 and express CD45 and CD150. ESC-HSCs express CD41 and CD150, lack CD34, and are heterogeneous for CD45. Finally, although CD48 was absent from all in vivo HSCs examined, ESC-HSCs were heterogeneous for the expression of this molecule. This unique phenotype signifies a developmentally immature population of cells with features of both primitive and mature HSC. The prospective fractionation of ESC-HSCs will facilitate studies of HSC maturation essential for normal functional engraftment in irradiated adults.