This study investigated the ability of nanoscale, biomimetic peptide amphiphile (PA) scaffolds inscribed with specific cellular adhesive ligands to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs) without osteogenic supplements. PA sequences were synthesized to mimic the native bone extracellular matrix (ECM), expressing different isolated ligands (i.e., RGDS, DGEA, KRSR). All PAs were presented as self-assembled two-dimensional coatings for the seeded hMSCs. Initial attachment results demonstrated that the different PAs could be individually recognized based on the incorporated adhesive ligands. Long-term studies assessed osteogenic differentiation up to 35 days. The RGDS-containing PA nanomatrix expressed significantly greater alkaline phosphatase activity, indicating the early promotion of osteogenic differentiation. A progressive shift toward osteogenic morphology and positive staining for mineral deposition provided further confirmation of the RGDS-containing PA nanomatrix. Overall, the PA nanomatrix clearly has great promise for directing the osteogenic differentiation of hMSCs without the aid of supplements by mimicking the native ECM, providing an adaptable environment that allows for different adhesive ligands to control cellular behaviors. This research model establishes the beginnings of a new versatile approach to regenerate bone tissues by closely following the principles of natural tissue formation. © 2009 American Chemical Society.