The forkhead box C2 (Foxc2) protein is a member of the family of winged helix/forkhead transcription factors. Foxc2-deficient mice display defective formation of the aortic arches, multiple craniofacial bones, and vertebral columns. To investigate the role of Foxc2 in osteoblast differentiation, DNA containing Foxc2 was transfected into the developing cranial suture mesenchymal cells by electroporation. Compared to the controls, alkaline phosphatase (ALP) and bone sialoprotein were expressed strongly in suture mesenchymal cells in the Foxc2 overexpressed calvaria. After Foxc2-siRNA transfection, ALP staining was rarely observed in the suture mesenchyme and adjacent parietal bone of the calvaria. Meanwhile, overexpression of Foxc2 increased protein levels of beta-catenin and stimulated TCF/LEF transcriptional activity. The protein kinase A inhibitor H-89 suppressed Foxc2-mediated increases in TCF/LEF transcriptional activity (-40%, P<0.01). In conclusion, our results demonstrated that Foxc2 stimulated osteoblast differentiation of mesenchymal cells and preosteoblasts. Activation of canonical Wnt-beta-catenin signals might be involved in the Foxc2-mediated stimulation of osteoblast differentiation.