Cues from the material to which a cell is adherent (e.g., adhesion ligand presentation, substrate elastic modulus) clearly influence the phenotype of differentiated cells. However, it is currently unclear if stem cells respond similarly to these cues. This study examined how the overall density and nanoscale organization of a model cell adhesion ligand (arginine-glycine-aspartic acid [RGD] containing peptide) presented from hydrogels of varying stiffness regulated the proliferation of a clonally derived stem cell line (D1 cells) and preosteoblasts (MC3T3-E1). While the growth rate of MC3T3-E1 preosteoblasts was responsive to nanoscale RGD ligand organization and substrate stiffness, the D1 stem cells were less sensitive to these cues in their uncommitted state. However, once the D1 cells were differentiated towards the osteoblast lineage, they became more responsive to these signals. These results demonstrate that the cell response to material cues is dependent on the stage of cell commitment or differentiation, and these findings will likely impact the design of biomaterials for tissue regeneration. ?? 2007 Wiley Periodicals, Inc. J Biomed Mater Res 2008