A cell-imprinted poly(dimethylsiloxane)/hydroxyapatite nanocomposite substrate was fabricated to engage topographical, mechanical, and chemical signals to stimulate and boost stem cell osteogenic differentiation. The physicochemical properties of the fabricated substrates, with nanoscale resolution of osteoblast morphology, were probed using a wide range of techniques including scanning electron microscopy, atomic force microscopy, dynamic mechanical thermal analysis, and water contact angle measurements. The osteogenic differentiation capacity of the cultured stem cells on these substrates was probed by alizarin red staining, ALP activity, osteocalcin measurements, and gene expression analysis. The outcomes revealed that the concurrent roles of the surface patterns and viscoelastic properties of the substrate provide the capability of directing stem cell differentiation toward osteogenic phenotypes. Besides the physical and mechanical effects, we found that the chemical signaling of osteoinductive hydroxyapatite nanoparticles, embedded in the nanocomposite substrates, could further improve and optimize stem cell osteogenic differentiation.