Introduction: Adipose tissue-derived mesenchymal stem cells (AdMSC) are capable of differentiating into endothelial cell lineages. Hypothesis: We evaluated the effect of culture-expanded human AdMSC on in vitro angiogenesis in the presence of shear stress (SS) or growth factors and compared the effect of angiogenesis with three different cell numbers of AdMSC in ischemic hind limb mouse model. Methods: AdMSC were isolated and cultured from human adipose tissue. Three or four passage of AdMSC were exposed to unidirectional laminar SS. Hindlimb ischemia was induced by ligating the femoral vessels of forty male BalbC/nude mice. With three different number of AdMSC treated group (1.0X106 cells/kg in LD group, 5.0X106 cells/kg in MD group, 1.0X107 cells/kg body weight in HD group) and media-treated control group, cells were directly injected into ischemic muscle one day after ligation. Using lentivirus-eGFP transfected AdMSC, the differentiation of injected AdMSC were evaluated. Results: Sphere formation during culture expansion of AdMSC was marked increased. In vitro, AdMSC could form tube formation in metrigel assay and expressed vW factor and KDR. Ac-LDL and UEA lection double-positive cells were considered as endothelial-like cell. SS rather than growth factor accelerated the increments of the expression of Flt-1, V-catherin, KDR as endothelial cell marker by RT-PCR and immunocytochemistry. AdMSC pretreated with SS had resistance to apoptotic effect of TNF-alpha analyzed by Annexin V expression using FACS. The laser Doppler perfusion index was significantly improved in AdMSC-treated group than in the control group (61.0% in LD group, 79.5% in MD group, and 97.5% in HD group; p<0.05) on day 28. Histological examination showed that AdMSC transplantation increased capillary density, compared with the control group (64.6% in LD group, 73.8% in MD group, and 79.3% in HD group; p<0.05). The effect of AdMSC was correlated with the number of transplanted cells. A few GFP-positive cells could be detected in ischemic muscle, and were also stained for CD31. Conclusion: Human AdMSC could be one of ideal adult stem cell source for clinical therapeutic angiogenesis in ischemic cardiovascular disease.