Embryonic Stem Cells (ESCs) are expected to show a stable euploid karyotype, but in the last decade (sub)chromosomal aberrations have been systematically described in these cell lines when maintained in vitro. Culture conditions and long-term culture have been traditionally proposed as possible factors involved in the acquisition of chromosomal abnormalities. Thus, we analyzed the chromosome constitution, the undifferentiated state and the functional pluripotency of three different mouse ESCs grown under the same culture conditions. Two cell lines were unstable from early passages, whereas the third one retained its chromosome integrity after long-term culture despite using enzymatic methods for cell disaggregation. Trisomy 8 and 11 were clonally selected in both unstable cell lines, which also showed a higher growth rate than our normal cell line and suffered morphological changes in colony shape with increasing passage number. Regardless of the length of culture or the chromosome instability, all cell lines preserved their differentiation potential. These results confirm that double trisomy 8 and 11 confers a growth advantage to the abnormal cells, but not at the expense of cell differentiation. The presence of chromosome instability, widely related to tumor development and cancer disease, highlights the risk of using pluripotent cells in regenerative medicine.