Objective:To establish an efficient and stable method for simultaneous isolation and culture of murine mesenchymal stem cells and endothelial progenitor cells from bone marrow. Methods:The mononuclear cells were isolated from murine bone marrow and cultured for 2 days. Then the adherent cells were cultured in LG-DMEM with 10% preselected FBS for MSC and the suspending cells were cultured in EGM-2 MV for EPC, respectively. Osteogenic and adipogenic induction was performed on MSCs. The percentage of MSCs was analyzed by flow cytometry(FCM). EPCs were stained with Dil-ac-LDL and FITC-UEA-1, and the expression of vWF and CD31 was also assessed by immunohistochemistry. Results:The early attached cells exhibited clonal morphology as early as 48 hours, which became 80% confluent within 1 week. MSCs could differentiate into osteocytes and adipocytes after induction. The expression of CD29,CD34,CD45 and CD90 were(93.86 ± 1.12)%,(0.48 ± 0.38)%,(1.89 ± 1.49)%,(94.11 ± 3.32)%, respectively. Cells cultured in EGM-2 MV became confluent after 2 weeks,(75.2 ± 4.5)% of which were double positive for Dil-ac-LDL and FITC-UEA-1 staining. The percentages of vWF+ and CD31+ cells were (55.7 ± 4.7)% and (52.5 ± 3.6)%, respectively. Conclusion:An efficient, stable and replicable method for simultaneous isolation and culture of MSCs and EPCs from a single mouse was established.