Cellular mechanical properties are implicated in numerous cell behaviors, but their involvement in cell differentiation process has remained unclear. Since mechanical interactions between chondrogenic cells and their surrounding environment heavily affect the maintenance of their differentiation phenotype, here, using a chondrogenic cell strain ATDC5, we evaluated cell mechanical properties (e.g., adhesive force and spring constant) and gene expression levels in differentiation culture. The adhesive force appeared to be affected by both cellular cytoskeletal and adhesive constructions. Treatment with Y27632, which accordingly inhibits actin polymerization, decreased the adhesive force while increased chondrogenic gene expressions, suggesting the both of them are interrelated via the mediation of actin cytoskeleton. However, the mechanical property did not represent chondrogenic differentiative stages as obviously as the biochemical characteristics. Meanwhile, interestingly, changes in cell distribution maps of the force in the differentiation process indicated that the cells have different levels of mechanical properties in the undifferentiated state, whereas they tend to converge when the differentiative stage is in a lull. These results reaffirm the cellular diversity during differentiation from a mechanical perspective and provide important information to the fields of generation and scaffold-based tissue regeneration, where cell-substrate adhesion plays a role.