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Embryonic stem (ES) cells have the potential to differentiate into every cell type in the body, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. Here, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-1+) mouse ES cells were exposed to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S phase and G2-M phase of the cell cycle than Flk-1+ ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, VE-cadherin, and PECAM-1, at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker SM-α-actin, the blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-1+ ES cells into the endothelial cell lineage rather than into other cell lineages.