Objective:To establish a culture method for primary mesenchymal stem cells from neonatal mouse calvaria. Methods:Mesenchymal stem cells were collected from neonatal mouse calvaria with collagenase. The morphological changes and proliferation of the cultured cells were observed. Cell circle stages were analyzed by flow cytometry. The expression of cell-surface antigens were detected by flow cytometry. The potential of the isolated cells to differentiate into osteogenic and adipogenic lineages was examined. The mineralization and the intracytoplasmic lipid were detected using alizarin red staining and oil red O staining,respectively. Differentiation abilities were further validated by RT-PCR analysis of osteoblast specific gene and adipocyte specific genes. Results:The cultured spindle-like cells showed highly homogeneous appearance with active proliferation. The cells were positive for CD29 and CD44, while negative for CD34 and CD45. Visible mineralized nodules and lipid droplets were observed with alizarin red staining and oil red staining,respectively. In addition,the mRNA expression of osteoblast specific gene and adipocyte specific genes increased obviously after induction. Conclusion:Our research successfully established a culture method for primary mesenchymal stem cells from neonatal mouse calvaria. This method might be important for further studies of mesenchymal stem cells.