Understanding the fundamental mechanisms that trigger deviation from the undifferentiated state of human induced pluripotent stem cells (hiPSCs) provides key strategies to maintain their undifferentiated state during cell expansion. We assessed deviation from the undifferentiated state in hiPSC colonies by measuring cell migration rates in colonies with deviation that were targeted by the end of culture, in a backward manner. Analyses of migration rates of single cells in colonies with deviation demonstrated that the distribution of migration rates at the region with occurrence of deviated cells had a broad or narrow range compared with those at the regions of undifferentiated cells. It was found that deviated cells in hiPSC colonies accidentally occurred consequent to the appearance of relatively fast or slow migrating cells at the peripheral or central region of colonies, reflecting disorders owing to cell migration anomalies in the hiPSC colony. Fluorescence microscopy for F-actin, paxillin, and E-cadherin clarified the localization of integrin-mediated and cadherin-mediated adhesions, introducing the concept that the occurrence and pattern of deviation in a colony were responsive to changes of cell migration in that colony. Furthermore, a major component of the nuclear lamina, laminA/C displayed a rim at the nuclear periphery in the regions with occurrence of deviated cells, concomitant with the actin cytoskeleton associated with integrin- and cadherin-mediated cell adhesion. These results showed that an anomaly of cell migration in hiPSC colonies led to the accidental appearance of deviated cells therein through alternation of the nuclear lamina and imbalance between cell cell and cell-substrate interactions. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.