Mesenchymal stem cells (MSCs) are multipotent stromal cells found in mesenchymal tissues. They are known to contribute to vascular tissue remodeling and repair, but the underlying biology is unclear. It is thought that they can release soluble cytokines which encourage angiogenesis and also that they can contribute to vessel formation by differentiating along endothelial and vascular smooth muscle cell (SMC) lineages(1). MSC differentiation into SMCs is not well understood, but interactions between MSCs and the extracellular matrix (ECM) are important. Differentiation can be tracked by the presence of specialised contractile proteins: Smooth muscle α-actin (SMA) and calponin-h1 during early differentiation; smoothelin-B during intermediate differentiation; and smooth muscle myosin heavy chain (SM MHC) during late differentiation. We investigated how interactions between MSCs and the ECM influence differentiation towards SMC phenotype by culturing MSCs on gelatin, fibronectin or fragments of the elastic matrix molecule fibrillin-1. Bone-marrow derived MSCs were cultured in vitro on surfaces pre-coated with gelatin, fibronectin, or integrin-binding fragments of fibrillin-1 (PF17, which is further divided into PF8 (binds integrin α5β1) and PF9 (binds integrin αvβ3(2)) for up to 14 days. MSCs were then analysed for SMC marker expression by immunofluorescence, western analysis and RT-PCR. MSCs cultured for 14 days on gelatin, fibronectin or PF17 displayed evidence of the late differentiation marker SM MHC on immunofluorescence microscopy. Culture of MSCs on gelatin or fibronectin led to mRNA expression of SMA, calponin-h1 and smoothelin-B. Culture of MSCs on different concentrations of PF17 for 7 days produced a PF17 concentration dependent increase in calponin-h1 protein expression (p<0.01, one-way ANOVA). However MSCs cultured on different concentrations of fibronectin displayed similar levels of both SMA and calponin-h1 protein expression. Culture of MSCs on PF17 or PF8 for 7 days induced similar expression of calponin-h1 whereas MSCs cultured on PF9 for 7 days expressed less calponin-h1. Previous studies into MSC differentiation towards SMC phenotype have focused on the role of transforming growth factor-β and cyclical stretch. I demonstrate that culture of MSCs on certain ECM molecules is sufficient to induce early, intermediate and late SMC marker expression. Furthermore, the novel finding that PF17, but not fibronectin, induces concentration-dependent expression of calponin-h1 suggests that there is key interplay between fibrillin-1 and MSC differentiation towards SMC phenotype. The decreased expression of calponin-h1 seen in MSCs cultured on PF9 compared to PF8 and PF17 suggests that specific integrin signaling may also be involved.