Abstract: AIM: To establish an efficient and stable method for simultaneous isolation, culture and differentiation of murine endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were isolated from murine bone marrow by density gradient (Histopaque-1077, Sigma) after differential centrifugation. The resulting cells were cultured and differentiated to endothelial cells (ECs) in EBM-2. The expressions of specific antigens (CD133, CD34, Flk-1 and CD31) on cell surface were analyzed by immunofluorescence and flow cytometer. Biological functions of endothelial cells were examined by the adsorption of ulex europaeus-agglutinin (UEA) labeled by fluorescein isothiocyanate (FITC) and DiI-ac-LDL internalization. Expression of vWF and CD31 of ECs differentiated from EPCs was also assessed by immunohistochemistry. RESULTS: Cells were obtained from mouse bone marrow by density gradient, and adhesive cells expressed CD133, CD34 and Flk-1 and formed clusters at the 4th day. At the 14th day, UEA adsorption was labeled by FITC and DiI-ac-LDL internalization were positive. Expression of CD133 in adhesive cells decreased, whereas CD31 increased. Positive ratios of CD34+, CD133+, Flk-1+ and CD31+ were (43.55±4.12)%, (18.29±2.56)%, (48.78±3.96)% and (78.89±6.38)%, respectively. After 3 weeks, they differentiated into mature ECs forming cobblestone monolayers. There was no difference in secretion of PGI2 between ECs from EPCs and ECs from mouse aorta. CONCLUSION: An efficient, stable and replicable method for simultaneous isolation and culture of MSCs and EPCs from a single mouse has been established.