Mechanoregulation of cell–extracellular matrix (ECM) interactions are crucial for dictating pluripotent stem cell differentiation. However, not all pluripotent cells respond homogeneously which results in heterogeneous cell populations. When cells, such as mouse epiblast stem cells (EpiSCs), are cultured in clusters, the heterogeneity effect during differentiation is even more pronounced. While past studies implicated variations in signaling pathways to be the root cause of heterogeneity, the biophysical aspects of differentiation have not been thoroughly considered. Here, we demonstrate that the heterogeneity of EpiSC differentiation arises from differences in the colony size and varying degrees of interactions between cells within the colonies and the ECM. Confocal imaging demonstrates that cells in the colony periphery established good contact with the surface while the cells in the colony center were separated by an average of 1–2 µm from the surface. Traction force measurements of the cells within the EpiSC colonies show that peripheral cells generate large tractions while the colony center cells do not. A finite element modeling of EpiSC colonies shows that tractions generated by the cells at the colony periphery lift off the colony center preventing the colony center from undergoing differentiation. Together, our results demonstrate a biophysical regulation of heterogeneous EpiSC colony differentiation.