Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development, but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1⁺) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain, and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 hours), the Flk-1⁺ ES cells significantly increased in cell density and became oriented perpendicular to the direction of strain. There were marked increases in the VSMC markers SM -actin and SM-myosin heavy chain (SM-MHC) at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell (EC) marker Flk-1 decreased, and there were no changes in the other EC markers Flt-1, VE-cadherin, and PECAM-1, the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor (PDGFR) kinase inhibitor AG1296 completely blocked the cyclic-strain-induced increase in cell density and VSMC marker expression. Cyclic strain (8%, 1 Hz) immediately caused phosphorylation of PDGFR, but neutralizing antibody against PDGF-BB did not block the PDGFR phosphorylation. These results suggest that cyclic strain activates PDGFR in a ligand-independent manner, and that the activation plays a critical role in VSMC differentiation from Flk-1⁺ ES cells.