The possibility of using multipotent adult bone marrow-derived mesenchymal stem cells (MSCs) for tissue-engineering applications hinges on the ability to predictably control their differentiation. Previously, we showed the osteogenic potential of adult bone marrow-derived MSCs cultured on thin films of poly(lactide-co-glycolide) (PLGA) depends in part on the identity of extracellular matrix (ECM) ligands initially deposited onto the material from serum in the culture medium. Here we have addressed the hypothesis that remodeling of the PLGA surface via the de novo synthesis of ECM proteins by the MSCs may also play an important role in governing their osteogenic differentiation. Supporting this hypothesis, increasing amounts of fibronectin and type-I collagen were synthesized and deposited onto thin-film PLGA substrates, whereas vitronectin levels diminished over a 28-day time course. Integrin expression profiles changed accordingly, with higher levels of alpha2beta1 and alpha5beta1 than alphavbeta3 at three different time points. The mitogen-activated protein kinase (MAPK) and phosphatidyl inositol-3-kinase (PI3K) pathways were also activated in MSCs cultured on these substrates, and their inhibition significantly inhibited osteogenic differentiation as assessed according to alkaline phosphatase activity and mineral deposition. These data indicate that initial ECM deposition, subsequent matrix remodeling, and corresponding integrin expression profiles influence osteogenesis in MSCs cultured on PLGA in part by engaging MAPK and PI3K signaling pathways. Understanding the mechanisms by which stem cells respond to different polymers will be critical in their eventual therapeutic use.