During embryogenesis, the hematopoietic program is specified from the mesodermal germ layer through the formation of hemangioblast. This precursor gives rise to a hemogenic endothelium that later on matures to generate primitive and definitive hematopoietic precursors. A lack of specific cell surface markers to identify cells with discrete developmental potential is a major hurdle in the quest to further understand the cellular and molecular program governing blood formation. In the present study, we identify CD40 and Icam2, two markers typically associated with the adult immunological compartment, as expressed at the earliest stages of blood specification both in vitro and in vivo. Using in vitro serum-free culture conditions that support the efficient and directed differentiation of embryonic stem cells, we show that the sequential expression of CD40 and Icam2 delineate a transition in the acquisition of the blood potential from hemangioblast to hemogenic endothelium leading to the formation of primitive and definitive hematopoietic progenitors. CD40 is transiently expressed at the onset of blood development and marks first the hemangioblast then the hemogenic endothelium but is no longer expressed on fully committed hematopoietic precursors within the fetal liver. In contrast, Icam2 is first expressed on the hemogenic endothelium and its expression persists on fetal liver hematopoietic progenitors. Taken together, our data identify novel cell surface markers allowing us to further refine our understanding of the events marking progressive hematopoietic commitment from the mesoderm germ layer.