In this study, we examined the signaling pathways activated by Wnt5a in endothelial differentiation of embryonic stem (ES) cells and the function of Wnt5a during vascular development. We first found that Wnt5a(-/-) mouse embryonic stem (mES) cells exhibited a defect in endothelial differentiation, which was rescued by addition of Wnt5a, suggesting that Wnt5a is required for endothelial differentiation of ES cells. Furthermore, a defect in the vascularization of the neural tissue was detected at embryonic day 14.5 in Wnt5a(-/-) mice, implicating Wnt5a in vascular development in vivo. Thus, we conclude that Wnt5a is involved in the endothelial differentiation of ES cells via both Wnt/beta-catenin and PKC signaling pathways and regulates embryonic vascular development.