Multipotent human mesenchymal stem cells (hMSCs) are uniquely suited for the growing field of regenerative medicine due to their ease of isolation, expansion, and transplantation. However, during ex vivo expansion necessary to obtain clinically relevant cell quantities, hMSCs undergo fundamental changes culminating in cellular senescence. The molecular changes as hMSCs transition into senescence have been well characterized, but few studies have focused on the mechanical properties that govern many processes necessary for therapeutic efficacy. We show that before detectable differences in classical senescence markers emerge, single-cell mechanical and cytoskeletal properties reveal a subpopulation of 'non-functioning' hMSCs that appears after even limited expansion. This subpopulation, characterized by a loss of dynamic cytoskeletal stiffening and morphological flexibility in response to chemotactic signals grows with extended culture resulting in overall decreased hMSC motility and ability to contract collagen gels. These changes were mitigated with cytoskeletal inhibition. Finally, a xenographic wound healing model was used to demonstrate that these in vitro differences correlate with decreased ability of hMSCs to aid in wound closure in vivo. These data illustrate the importance of analyzing not only the molecular markers, but also mechanical markers of hMSCs as they are investigated for potential therapeutics.