The capacity of the yolk sac to generate multilineage, adult-type hematopoiesis was investigated in vivo using vascular endothelial-cadherin deficient embryos. In these mutants, the yolk sac is not connected to the vasculature of the embryo and therefore all hematopoietic activity detected therein is intrinsic to the yolk sac and not derived from intraembryonic sources. At embryonic days 9.5 and 10.5, the yolk sac contains blood cells from the first wave of hematopoiesis, i.e. primitive erythrocytes and monocytes, but also multipotent progenitors from definitive hematopoiesis and a few granulocytes. Reverse transcription-polymerase chain reaction analysis revealed expression of specific genes of all lineages except lymphoid cells. Moreover, hematopoietic colony assays showed the existence of committed progenitors of the second wave of embryonic hematopoiesis, namely for definitive erythrocytes, megakaryocytes, granulocytes and monocytes. Conversely, the number of lymphocytes after lymphoid culture was insignificant. Our data provide evidence for multilineage hematopoiesis (but not lymphopoiesis) in the yolk sac in the absence of seeding from the embryo. The small number of definitive mature blood cells indicates however that the yolk sac is not an effective environment for the terminal differentiation of committed progenitors from the second wave of hematopoiesis.