Embryonic stem cells (ESCs) overexpressing the vascular endothelial growth factor (VEGF) improve cardiac function in mouse models of myocardial ischemia and infarction by mechanisms that are poorly understood. Here we studied the effects of VEGF on cardiomyocyte differentiation of mouse ESCs in vitro. We used flow cytometry to determine the expression of -myosin heavy chain (-MHC), cardiac troponin I (cTn-I), and Nkx2.5 in differentiated ESCs. VEGF (20 ng/ml) significantly enhanced -MHC, cTn-I and Nkx2.5 expression in differentiated ESCs. Western blot analysis confirmed these findings. We found that VEGF receptors Flt-1 and Flk-1 expression increased during ESC differentiation. Antibodies against Flk-1 totally blocked and against Flt-1 partially blocked VEGF-induced NKx2.5 positive stained cells. The ERK inhibitor, PD098059, abolished VEGF-induced cardiomyocyte differentiation of ESCs. Our results suggest that VEGF promotes cardiomyocyte differentiation predominantly by ERK-mediated Flk-1 activation and, to a lesser extent, by Flt-1 activation. These findings may be of significance for stem cell and growth factor therapies to regenerate failing cardiomyocytes.