The anatomical location of lymphocyte ontogeny in the developing mouse embryo remains controversial. To define the site that can generate lymphocytes de novo, the intraembryonic splanchnopleura (SP) and extraembryonic yolk sac (YS) at 8.5 days postcoitum, when systemic circulation is not established, were investigated. The results indicated that in standard colony forming assay, the cells from both splanchnopleura and yolk sac formed typical myeloerythroid colonies, but their types were distinct. When cocultured with the OP9, the splanchnopleura produced B cells expressing B220, CD19 and surface IgM. Using a three-step culture protocols with the OP9 expressing Delta-like 1 as feeders, the splanchnopleura produced immature T precursor cells (CD44-/CD25+) and more mature single positive T cells (CD4+/CD8-) after 16 days of incubation. However, the yolk sac failed to generate B and T lymphocytes under identical conditions. It is concluded that prior to linked embryonic circulation, the splanchnopleura other than the yolk sac had robust lymphoid potential in vitro. In the future, more reliable evidence from novel model animals will ultimately delineate the embryonic origin of lymphocytes in vivo.