Embryonic stem cells differentiate blood vessel-like structures as well as smooth muscle cells. The interaction between endothelial cell and smooth muscle differentiation is so far not sufficiently investigated. In the present study it is shown that the mRNA expression of the endothelial cell markers CD31 and VE-cadherin as well as the smooth muscle markers calponin, caldesmon, a- smooth muscle actin and SM22a were upregulated within 8 days of differentiation. Immunohistochemical analysis revealed that CD31-positive endothelial cell structures were partially lined by smooth muscle cells, indicating that true blood vessel-like structures were differentiated. To investigate the effects of growth factors on endothelial/smooth muscle cell differentiation, embryoid bodies derived from embryonic stem cells were incubated with the growth factors angiopoietin-1 and ?2, vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF BB), and transforming growth factor-ß1 (TGF-ß1), and mRNA expression of endothelial versus smooth muscle cell markers was investigated. Our data demonstrate that the spontaneous differentiation of endothelial cell/smooth muscle differentiation from embryonic stem cells can be selectively directed by external addition of pro-angiogenic growth factors.