The role of mechanical forces in regulation of skeletal multi-potent cell differentiation, and consequently bone growth and skeletal disease, was investigated. A novel calvaria explant culture model was created to investigate mechanical strain effects on multi-potent suture cell differentiation and bone growth in the etiology of craniosyntostosis. Controlled 1.2% cyclic strain was applied to murine calvarial explants ex vivo for thirty minutes a day over a three-week period and alteration of osteoblast marker expression, osteoid synthesis, and suture fusion characterized. To precisely probe mechanical strain effects bn multi-potent cell differentiation and investigate the modulation of strain effect by cell culture environmental factors, bone marrow stromal cells (BMSC) were subject to controlled substrate deformation in vitro. BMSC were cultured on flexible silicone substrates that were ultraviolet irradiated or coated with fibronectin and subject to cyclic tensile strain (1.2% vs. 10%) for one hour a day, over a sixweek period, and effects on proliferation and phenotypic gene and matrix expression investigated. In the calvaria studies, ex vivo cyclic strain was found to promote murine suture cell differentiation and bone production at the suture, thereby decreasing sagittal suture patency, indicative of craniosynostosis. These effects were strongly mediated by soluble paracrine acting factors produced in response to strain. In HMSC studies, FN inhibited osteogenesis while 1.2% strain augmented osteogenesis on unmodified and irradiated substrates over six weeks culture. Both strain and substrate modification served to shift the balance between adipogenic vs. osteogenic differentiation through mitigation of cell clustering. Substrate modification also altered strain regulation of HMSC proliferation. FN was found to promote expression of chondrocyte specific gene markers, but long-term two-dimensional culture conditions with osteogenic medium supplements were not conducive to cartilage matrix synthesis as evidenced by adipogenesis. Thus, low magnitude strain (1.2%) possessed osteogenic effects in calvaria tissue explants and isolated HMSC in vitro. These results have application in basic BMSC biology, modulation of skeletal tissue regeneration, and tissue engineering therapies. Results may be applied to control BMSC differentiation into desired tissue phenotypes and common signaling pathways implicated by mechanical loading and known genetic disorders of skeletogenesis may be targeted for therapeutic intervention.