Multi-potent adult mesenchymal stem cells (MSCs) derived from bone marrow have therapeutic potential for bone diseases and regenerative medicine. However, an intrinsic heterogeneity in their phenotype, which in turn results in various differentiation potentials, makes it difficult to predict the response of these cells. The aim of this study is to investigate initial cell–surface interactions of human MSCs on modified titanium alloys. Gold nanoparticles deposited on β-type Ti–40Nb alloys by block copolymer micelle nanolithography served as nanotopographical cues as well as specific binding sites for the immobilization of thiolated peptides present in several extracellular matrix proteins. MSC heterogeneity persists on polished and nanopatterned Ti–40Nb samples. However, cell heterogeneity and donor variability decreased upon functionalization of the gold nanoparticles with cyclic RGD peptides. In particular, the number of large cells significantly decreased after 24 h owing to the arrangement of cell anchorage sites, rather than peptide specificity. However, the size and number of integrin-mediated adhesion clusters increased in the presence of the integrin-binding peptide (cRGDfK) compared with the control peptide (cRADfK). These results suggest that the use of integrin ligands in defined patterns could improve MSC-material interactions, not only by regulating cell adhesion locally, but also by reducing population heterogeneity.