The murine yolk sac, being the first site of embryonic blood cell production, has long been theorized to contain the migrating hematopoietic stem cells (HSC) that seed the liver and initiate hematopoiesis on day 10.0 postcoitus (pc). However, it remains controversial whether yolk sac cells isolated before day 11.0 pc possess any long-term repopulating HSC activity upon transplantation into adult recipient mice. We hypothesized that failure to demonstrate engraftment of day <11.0 yolk sac cells in adult hosts may result from an inability of yolk sac cells to home to the active adult hematopoietic sites (spleen and bone marrow). In the present studies, we transplanted yolk sac cells into conditioned newborn mice in whom the liver, as well as the spleen and bone marrow, concomitantly function as a site of blood cell formation. We report that yolk sac cells isolated from day 9.0 pc embryos provide long-term multilineage reconstitution for at least 11 months in primary conditioned newborn mice and for at least 6 months in secondary recipients. Donor yolk sac HSC progeny repopulated mature peripheral blood, thymus, spleen, and bone marrow lymphoid, myeloid, and erythroid compartments. Thus, day 9.0 pc yolk sac HSC can contribute to definitive multilineage hematopoiesis in transplanted recipients. Determination of HSC activity in the day 9.0 pc murine yolk sac suggests that yolk sac HSC are available to seed the liver on day 10.0 pc when definitive hematopoiesis is initiated.