Embryonic stem (ES) cells and induced pluripotent stem cells recently attracted attention as materials and treatment methods for tissue regeneration. However, molecular mechanisms which lead to the cell differentiation into various specialized cells aren’t fully understood. It has been reported that the addition of vascular endothelial growth factor (VEGF) to ES cell cultures promotes endothelial cell (EC) differentiation, whereas mural cells, including smooth muscle cells and pericytes, are induced by platelet-derived growth factor (PDGF). In this study, we examined the effects of shear stress or uni-axial stretching in ES differentiation. Shear stress markedly increased the expression of EC-specific markers, and the shear-induced differentiation into ECs were completely blocked by the VEGF receptor kinase inhibitor SU1498. Whereas, stretch significantly increased the mural cell markers, and the stretch-induced increases were completely blocked by the PDGF receptor inhibitor AG1296. These results indicate that hemodynamic forces selectively promote ES cell differentiation via mechanotransduction pathways.