The therapeutic use of embryonic stem (ES)-derived cells is restricted by a risk of teratoma formation. To test the hypothesis that some cells with pluripotency characteristics remain following the differentiation of embryoid bodies (EB) into monolayer cells, we transformed mouse ES cells using constructs comprised of the mTert promoter coupled to green fluorescent protein. In this manner, EBs could be identified as showing gradually diminishing expression of the fluorescent marker as a consequence of differentiation. After 2 weeks of incubation, however, small groups of fluorescent cells remained in the differentiated monolayer. When these cells were isolated, cultured and expanded under ES cell culture conditions, they recovered their ES cell morphology (herein denoted ES-EB). We found by immunocytochemistry, reverse transcription-PCR and bisulphite analysis that despite the fact that some of these ES-EB lost their capacity to express some pluripotency markers characteristic of ES cells and undergo the epigenetic modification (hypo-methylation) of some retrotransposons (RT), after several passages in ES media, the cell colonies recovered their capacity for both pluripotency marker expression and RT methylation. Furthermore, when assessed for their ability to form chimeras, most ES-EB lines were unable to do so, although they recovered this potential for chimera production after some passages in ES cell media. Our results highlight the need for specific screening of differentiated cells before their therapeutic use and indicate that under adequate culture conditions, cells that loose their potential for expressing key markers of pluripotency can recover this fundamental embryonic stem cell property.