Mechanical forces transduced to the nucleus that affect intranuclear organization are critical regulators for directing the differentiation of mesenchymal stem cells (MSCs). The supraphysiological dose of a mechanical cue, which is from the stiff surface of the substrate like standard tissue culture polystyrene, biases the MSCs toward osteogenic differentiation with irreversible nuclear retention of an osteogenic master transcription factor, RUNX2. To study the mechanism underlying the loss of lineage plasticity and robustness of the irreversible osteogenic differentiation in human MSCs due to the mechanotransduction, we investigated how RUNX2 was retained in the nucleus focusing on the dynamic shuttling of a mechanotransducer, yes-associated protein (YAP), intranuclear chromatin organization, and their regulation by the actin cytoskeleton using pharmaceutical inhibitors to perturb the mechanical properties of chromatin and actin cytoskeleton. YAP and RUNX2 were localized in the nucleus of MSCs during the expansion culture on the polystyrene surfaces followed by 24 hours culture on the glass surface, at the time point just before the inhibitor treatments. The nuclear RUNX2 was diffused into the cytoplasm by chromatin decondensation through the inhibition of histone deacetylase. In contrast, RUNX2 localization was unaffected from the perturbations to the actin organization through the inhibition of actin polymerization and that of Rho kinase activity under the condition that chromatin condensation was maintained, although YAP was released from the nucleus to the cytoplasm. These findings suggest that RUNX2 is stably retained in the nucleus by its engagement with specific DNA and/or nucleoskeletal structures coexisting with the robustly condensed chromatin, in the MSCs biased toward an irreversible osteogenic differentiation.