Embryonic stem (ES) cells have the ability to maintain pluripotency and self-renewal during in vitro maintenance, which is a key to their clinical applications. ES cell quality has been widely evaluated through determination of their specific genetic and epigenetic profiles. The hypothesis of this study is that genetic stability in repetitive sequences located near key genes involved in pluripotency, self-renewal, differentiation, chromatin assembly, and imprinting could be a signal for adaptation of the ES cell in vitro. Instability in specific repetitive sequences is present and increases during ES cell passages. ES cells displayed significant mean frequencies of instability in twelve markers out of 64 related to pluripotency (OCT4, D1S551), early differentiation (G60405, D18S63, and D1S468), chromatin assembly (D22S447, D6S2252, D10S529, and HISTB2), and imprinting (GRB10-promoter, D2S144, and IGF2- promoter). Interestingly, instability was different between H1 and H7 ES cell lines. In summary, these results suggest that instability in tandem repeat sequences located near early embryonic developmental genes is associated with failure of ES cell pluripotency and self-renewal maintenance over consecutive culture passages. These results suggest that instability determination is a potential indicator of gene deregulation and epigenetic modification that involves chromatin modification and imprint establishment during ES cell culture. Finally, instability in specific genes could be a signal that contributes to adaptation of ES cells to in vitro culture or could be the switch that initiates early cell specialization in vitro.