With the multi-directional differentiation potential such as osteogenic differentiation, chondrogenic differentiation and adipogenic differentiation, mesenchymal stem cells (MSCs) have been widely used in basic research and clinical applications. The differentiation potential of MSCs is altered during senescence. Osteogenic differentiation potential decreases, while the lipogenic differentiation potential increases in aging MSCs. Changes in differentiation potential of MSCs during senescence are accompanied with cell physical heterogeneity variation (cell size, cell stiffness and nucleoplasmic ratio). Studies have shown that changes in physical heterogeneity of stem cells may be a key factor leading to the differences in differentiation potential of MSCs. Therefore, studies on physical heterogeneity variation of MSCs during senescence will provide a new research direction in fate prediction of stem cell. In this review, the effects of physical heterogeneity variation on differentiation potential of MSCs were summarized, and the corresponding mechanism was also discussed.