Background-Pericytes represent a unique subtype of microvessel-residing perivascular cells with diverse angiogenic functions and multilineage developmental features of mesenchymal stem cells. Although various protocols for derivation of endothelial and/or smooth muscle cells from human pluripotent stem cells (hPSC, either embryonic or induced) have been described, the emergence of pericytes in the course of hPSC maturation has not yet been elucidated. Methods and Results-We found that during hPSC development, spontaneously differentiating embryoid bodies give rise to CD105 ϩ CD90 ϩ CD73 ϩ CD31 Ϫ multipotent clonogenic mesodermal precursors, which can be isolated and efficiently expanded. Isolated and propagated cells expressed characteristic pericytic markers, including CD146, NG2, and platelet-derived growth factor receptor ␤, but not the smooth muscle cell marker ␣-smooth muscle actin. Coimplantation of hPSC-derived endothelial cells with pericytes resulted in functional and rapid anastomosis to the murine vasculature. Administration of pericytes into immunodeficient mice with limb ischemia promoted significant vascular and muscle regeneration. At day 21 after transplantation, recruited hPSC pericytes were found incorporated into recovered muscle and vasculature. Conclusions-Derivation of vasculogenic and multipotent pericytes from hPSC can be used for the development of vasculogenic models using multiple vasculogenic cell types for basic research and drug screening and can contribute to angiogenic regenerative medicine. (Circulation. 2012;125:87-99.)Key Words: pluripotent stem cells Ⅲ ischemia Ⅲ mesenchymal stem cells Ⅲ pericytes Ⅲ vasculature I n adult tissues, the majority of blood vessels are composed of 3 layers including a luminal inner monolayer of endothelial cells (EC), tunica intimae, an intermediate muscular layer, tunica media, of smooth muscle cells (SMC) and an outer layer of fibroblast-like adventitial cells and connective tissue components, tunica adventitia. Microvessels, including capillaries, precapillary arterioles, postcapillary venues, and collecting venules are composed of internal endothelial layer surrounded by outer coverage of pericytes (also known as Rouget cells or mural cells). 1 Both perivascular SMC and pericytes have been shown to function as critical regulators of vascular development, stabilization, maturation, and remodeling mediated by transforming growth factor ␤ (TGF-␤), platelet-derived growth factor B, or angiopoietin-1. 2,3 Although related in function and anatomic localization, pericytes can be distinguished from SMC on the basis of their characteristic morphology and specific cell marker expression: Whereas SMC form a separate layer of the tunica media in blood vessels, pericytes are physically embedded within the endothelial basement membrane to promote mutual communication with the underlying endothelium. 4 In addition, SMC and the majority of pericytes in multiple human and murine tissue types express ␣-smooth muscle actin (␣-SMA), which is involved in regulatio...