In gastrulating embryos, various types of cells are generated before differentiation into specific lineages. The mesoderm of the gastrulating mouse embryo represents a group of such intermediate cells. PDGF receptor alpha (PDGFRα), c‐Kit and fetal liver kinase 1 (Flk1) are expressed in distinctive mesodermal derivatives of post‐gastrulation embryos. Their expressions during gastrulation were examined by whole mount immunostaining with monoclonal antibodies against these three receptors. The antibodies stained different mesodermal subsets in gastrulating embryos. Flow cytometry of head fold stage embryos revealed that Flk1⁺ mesodermal cells could be further classified by the level of c‐Kit expression. To examine the possibility that hematopoietic cell differentiation is initiated from the Flk1⁺ mesoderm, embryonic stem (ES) cells were cultured on the OP9 or PA6 stromal cell layer; the former but not the latter supported in vitro hematopoiesis from ES cells. Flk1⁺ cells were detected only on the OP9 cell layer from day 3 of differentiation before the appearance of hematopoietic cells. Thus, Flk1⁺ cells will be required for in vitro ES cell differentiation into hematopoietic cells. The results suggest that these three receptor tyrosine kinases will be useful for defining and sorting subsets of mesodermal cells from embryos or in vitro cultured ES cells.