Introduction: Non-syndromic craniosynostosis is poorly understood and the molecular mechanisms are not known. We hypothesized that cells in fused suture express higher levels of osteogenic genes and produce paracrine factors that stimulate mesenchymal stem cells (MSCs) to differentiate into osteoblasts. To test this, we compared osteogenesis-related signaling pathways in cells isolated from normal bone (NB) and patent (PAT) and fused (FUS) sutures from human craniosynostosis patients. Methods: Osteoblasts isolated from NB, PAT, or FUS from 3 metopic (MET) or 3 lambdoid (LAM) craniosynostosis cases were characterized by responses of confluent cultures to treatment with 1α,25dihydroxyvitaminD3 (1,25D) for 24h. Cell number, alkaline phosphatase specific activity (ALP), and osteocalcin were measured. Expression of Wnts, BMPs, and integrins was measured by real-time qPCR. Cells were co-cultured with human MSCs. On d7, the osteoblasts were removed and cell number, ALP, and secreted factors analyzed. Data are meanSEM (n=6/condition, ANOVA and post-hoc Bonferroni's Student's t-test). Results: MET-FUS and LAM-FUS osteoblasts had higher ALP and osteocalcin and were more sensitive to 1,25D, than NB or PAT cells. For MET-FUS osteoblasts, DKK1 mRNA was lower and ITGA5, ITGB1, and βcatenin (CTNNB) higher than NB and PAT osteoblasts. In LAM-FUS osteoblasts, BMP4, FGF2, VDR and ITGB1 were up-regulated and DKK1 down-regulated in comparison to NB and PAT cells. LAM-PAT cells had higher CTNNB, LRP5, DKK2, ITGB1, and ITGA5 and lower RUNX2 than LAM-FUS cells. HMSCs co-cultured with MET-FUS or LAM-FUS osteoblasts had increased ALP, osteocalcin, osteoprotegerin, VEGF, TGFβ1, BMP2, and BMP4 in comparison to those with NB or PAT cells. Conclusion: Cells isolated from fused sutures in non-syndromic craniosynostosis present a more differentiated osteogenic phenotype and are more responsive to 1,25D3. They produce an osteogenic environment that induces osteoblastic differentiation in HMSCs. Candidate Wnt and integrin family genes were identified that may mediate suture fusion in non-syndromic craniosynostosis.