The microenvironment of bone marrow-derived human mesenchymal stem cells (hMSCs) strictly regulates their self-renewal and differentiation. Culturing these cells ex vivo leads to a rapid expansion followed by senescence, which is characterized by a lack of proliferation and differentiation. In this study, 250-Pa polyacrylamide gels, which mimics the elasticity of bone marrow and fat tissues, were coated with a mixture of collagen type 1 and fibronectin. When hMSCs were seeded sparsely on these gels, they halted progression through the cell cycle despite the presence of serum, but when presented with a stiff substrate, these non-proliferative cells reentered the cell cycle. Non-proliferative hMSCs on 250-Pa gels also exhibited the capability to differentiate into adipocytes when cultured in adipogenic induction medium or into osteoblasts if transferred to a stiff substrate and incubated with osteoblast induction medium. These results demonstrate that hMSCs on 250-Pa gels are quiescent but competent to resume proliferation or initiate terminal differentiation with appropriate cues. These observations suggest that mechanical signals from the elasticity of the extracellular matrix may be one of the factors that enable the bone marrow niche to maintain MSCs as a reservoir for a long period.