The differentiation of stem cells can be modulated by physical factors such as the micro- and nano-topography of the extracellular matrix. One important goal in stem cell research is to understand the concept that directs differentiation into a specific cell lineage in the nanoscale environment. Here, we demonstrate that such paths exist by controlling only the micro- and nano-topography of polymer surfaces. Altering the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts, in the absence of osteogenic medium. Small (10 nm) depth patterns promoted cell adhesion without noticeable differentiation, whereas larger depth patterns (100 nm) elicited a collective cell organization, which induced selective differentiation into osteoblast-like cells. This latter response was dictated by stress through focal-adhesion-induced reorganization of F-actin filaments. The results have significant implications for understanding the architectural effects of the in vivo microenvironment and also for the therapeutic use of stem cells.