Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to non-mineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration. Biotechnol. Bioeng. 2011;108: 2727–2735. © 2011 Wiley Periodicals, Inc.