Although information about the development of primitive and definitive hematopoiesis has been elucidated in murine embryos and embryonic stem (ES)cells, there have been few in vitro studies of these processes in primates. In this study, we investigated hematopoietic differentiation from cynomolgus monkey ES cells grown on OP9, a stromal cell line deficient in macrophage colony-stimulating factor. Primitive erythrocytes (EryP) and definitive erythrocytes (EryD) developed sequentially from ES cells in the culture system; this was confirmed by immunostaining and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of embryonic, fetal and adult globin genes. EryP were detected on day 8 without exogenous erythropoietin (EPO), whereas EryD appeared on day 16 and had an indispensable requirement for exogenous EPO. RT-PCR analysis of the cultures revealed a sequential expression of genes associated with primitive and definitive hematopoietic development that was equivalent to that seen during primate ontogeny in vivo. Vascular endothelial growth factor (VEGF) increased, in a dose-dependent manner, not only the number of floating hematopoietic cells,but also the number of adherent hematopoietic cell clusters containing CD34-positive immature progenitors. In colony assays, exogenous VEGF also had a dose-dependent stimulatory effect on the generation of primitive erythroid colonies. More efficient primitive and definitive erythropoiesis was induced by re-plating sorted CD34-positive cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of hematopoietic development in primates.