BACKGROUND:: Research approaches and measurement index regarding osteogenic differentiation of bone marrow mesenchymal stem cells in vitro are not fully considered at home and abroad. OBJECTIVE:: To establish and consummate a set of methods of isolating, culturing and identifying human bone marrow-derived mesenchymal stem cells (hBMSCs) and investigate their osteogenic differentiation ability in vitro. METHODS:: hBMSCs were isolated using the method of density gradient centrifugation. The cell surface phenotypes of BMSCs were identified by flow cytometry. Passage 3 hBMSCs were induced by osteogenic culture medium for 2-3 weeks. RESULTS AND CONCLUSION:: The primary cultured hBMSCs grew and proliferated vigorously after subculture. Passage 3 hBMSCs were positive for CD44, CD73, CD90 and were negative for CD34. After osteogenic induction, alkaline phosphatase activity was increased and cells were stained positive by Gomori, Von kossa and alizarin red. RT-PCR results showed the gene expression of collagen I, alkaline phosphatase, osteocalcin, bone sialoprotein, osteopotin and osteonectin. This confirms that the primary hBMSCs can differentiate into osteoblasts in vitro successfully. These findings suggest that a stable, mature method of isolating, culturing and amplifying hBMSCs has been established.Liu W, Liu M, Zhu JS, Hao HW, Dong NZ, Zhou HB. In vitro culture, identification and osteogenic differentiation of human bone marrow mesenchymal stem cells.Zhongguo Zuzhi Gongcheng Yanjiu. 2012;16(14): 2515-2519. [http://www.crter.cn http://en.zglckf.com]