The multi-lineage differentiation capacity of human mesenchymal stem cells (hMSCs) enables its potential for tissue engineering and regenerative medicine. Master transcription factors play a key role during development, differentiation, homeostasis and disease pathology. RUNX2 is the master transcription factor for bone development, and it regulates several important signaling pathways during chondrogenic and osteogenic differentiation of hMSCs. However, modulation of RUNX2 activity during hMSC differentiation into various lineages is not yet fully described. We differentiated hMSCs into chondro-, osteo-, and adipogenic lineages and studied RUNX2 protein dynamics using Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP) at different time points. The TF-FRAP method can capture the dynamic changes of RUNX2 protein mobility at the single cell level resolution, and cluster analysis shows how RUNX2 dynamics change at subpopulation level in proliferating and differentiating hMSCs. Our data show that although whole hMSC population is exposed to differentiation stimuli, some subpopulations in hMSCs do not respond to environmental cues.