Interactions with the extracellular matrix play important roles in regulating the phenotype and activity of differentiated articular chondrocytes; however, the influences of integrin-mediated adhesion on the chondrogenesis of mesenchymal progenitors remain unclear. In the present study, agarose hydrogels were modified with synthetic peptides containing the arginine-glycine-aspartic acid (RGD) motif to investigate the effects of integrin-mediated adhesion and cytoskeletal organization on the chondrogenesis of bone marrow stromal cells (BMSCs) within a three-dimensional culture environment. Interactions with the RGD-modified hydrogels promoted BMSC spreading in a density-dependent manner and involved αvβ3 integrin receptors. When cultured with the chondrogenic supplements, TGF-β1 and dexamethasone, adhesion to the RGD sequence inhibited the stimulation of sulfated-glycosaminoglycan (sGAG) production in a RGD density-dependent manner, and this inhibition could be blocked by disrupting the F-actin cytoskeleton with cytochalasin D. In addition, interactions with the RGD-modified gels promoted cell migration and aggrecanase-mediated release of sGAG to the media. While adhesion to the RGD sequence inhibited BMSC chondrogenesis in the presence of TGF-β1 and dexamethasone, osteocalcin and collagen I gene expression and alkaline phosphatase activity were enhanced by RGD interactions in the presence of serum-supplemented medium. Overall, the results of this study demonstrate that integrin-mediated adhesion within a three-dimensional environment inhibits BMSC chondrogenesis through actin cytoskeleton interactions. Furthermore, the effects of RGD-adhesion on mesenchymal differentiation are lineage-specific and depend on the biochemical composition of the cellular microenvironment. J. Cell. Physiol. 217: 145–154, 2008. © 2008 Wiley-Liss, Inc.