Restoration of the vascular supply to ischemic tissues is of high clinical relevance, and proangiogenic therapies aim to reduce morbidity and mortality rates associated with the onset of cardiovascular disease. Stem cell therapy has been proposed as a potentially useful proangiogenic therapy. Mesenchymal stem cells (MSCs) have been shown to be proangiogenic and produce a number of cytokines involved in vessel development and maturation. Preclinical studies have reported increased angiogenesis after MSC delivery to the heart, and similar outcomes have been reported in recent clinical trials. Stem-cell-mediated neovascularization has been augmented by genetic modification with overexpression of angiogenic cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor, showing promising results. In this study we aimed to enhance the proangiogenic capability of MSCs. MSCs were genetically modified to overexpress a versatile molecule, Ephrin-B2, involved in tissue morphogenesis and vascular development to enhance inherent neovascularization potential. Using nucleofection, Ephrin-B2 was transiently overexpressed on the cell surface of MSCs to recapitulate embryonic signaling and promote neovascularization. Ephrin-B2-expressing MSCs adopted an early endothelial phenotype under endothelial cell culture conditions increasing expression of von Willebrand factor and VEGF-Receptor 2. The cells had an increased ability to form vessel-like structures, produce VEGF, and incorporate into newly formed endothelial cell structures. These data indicate that MSCs expressing Ephrin-B2 represent a novel proangiogenic cell source to promote neovascularization in ischemic tissues.