Human bone marrow-derived mesenchymal stem cell (hMSCs) function depends on chemical factors and also on the physical cues of the microenvironmental niche. Here, this physical microenvironment is recapitulated with controlled modes of mechanical strain applied to substrata containing three-dimensional features in order to analyze the effects on cell morphology, focal adhesion distribution, and gene expression. 10% strain at 1 Hz is delivered for 48h to hMSCs cultured on flat surfaces, or on substrata with 15 μm-high microtopographic posts spaced 75 μm apart. Adding strain to microtopography produced stable semicircular focal adhesions, and actin spanning from post to post. Strain dominated over microtopography for expression of genes for the cytoskeleton (caldesmon-1 and calponin 3), cell adhesion (integrin-α2, vinculin, and paxillin), and extracellular matrix remodeling (MMP13) (P<0.05). Overall, attention to external mechanical stimuli is necessary for optimizing the stem cell niche for regenerative medicine.