The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.