Emergence of hemopoietic stem cells in the mammalian embryo has yet to be definitively allocated. Previously, we detected multipotent hemopoietic precursors in the region surrounding the dorsal aorta (paraaortic splanchnopleura) beginning at 8.5 days postcoitum (dpc). However, as circulation is already established, it remained unclear whether hemopoietic precursors arise in situ or are blood-delivered. By adding an organotypic step to our former culture system, we now detect lymphocyte and multipotent myeloid precursors from the intraembryonic splanchnopleura as early as 7.5 dpc. Under identical conditions, yolk sacs from the same embryos are unable to generate lymphoid progeny and have a reduced potential for myeloid differentiation and maintenance. Thus, if isolated before circulation, the yolk sac does not produce multipotent precursors and therefore does not contribute to definitive hemopoiesis in the mouse.