Self-assembled monolayer substrates containing tethered orthogonal concentration profiles of GRGDS and BMP-2 peptides are shown to synergistically accelerate the proliferation and osteoblastic differentiation of human mesenchymal stem cell (hMSC) populations in vitro without the use of osteogenic additives. Concurrently, the single peptide gradient controls (RGD or BMP-2 only) were found to induce significantly different proliferation and differentiation behavior from the orthogonal substrates. hMSC cells were individually isolated for qPCR at specified points along the gradients using laser capture microdissection. Bone sialoprotein (BSP) and Runt-related transcription factor 2 (Runx2) qPCR data corresponded spatially and temporally to protein marker data obtained from immunofluorescent imaging tracking the differentiation process. Genomic and protein data at high concentrations of both BMP-2 (25 pmol/cm2) and GRGDS (71~83 pmol/cm2) were shown to have a cooperative acceleration on the hMSC differentiation timeline relative to the individual peptide concentrations. These data highlight the utility of the orthogonal gradient approach to help identify the synergistic concentrations of peptides and growth factors that can be advanced in translationally relevant systems.