Mouse embryonic and induced pluripotent stem cells can be maintained in a pluripotent state, e.g. by cultivation in serum-containing medium supplemented with leukemia inhibitory factor (LIF). Expression of markers such as the transcription factors Oct-4 and Nanog or the surface carbohydrate SSEA-1 are mostly used to characterize pluripotency of mouse cells on a molecular level. Nevertheless, expression dynamics of these markers are rather low, leaving transcript / protein expression detectable even several days after a differentiation-inducing LIF removal. Therefore, these markers are limited in their potential to discriminate between pluripotent and early differentiated embryonic stem cells. A cell surface marker, absent on undifferentiated cells and rapidly upregulated upon early differentiation would be a valuable tool for cell separation approaches aiming at pluripotent stem cell isolation. In order to identify such an epitope we have analyzed the expression dynamics of different cell surface markers during mESC differentiation under various culture conditions. Using an in-house generated antibody, we have identifed a dynamically regulated surface marker, which is expressed in a small subpopulation of mESC (app. 10%) under standard cultivation conditions (FCS, LIF) and strongly upregulated to app. 60% during the first days of differentiation. Next we established a magnetic cell sorting (MACS) procedure to remove these early differentiated cells. On a morphological basis, especially fibroblast-like cells were efficiently depleted. Molecular characterization of the enriched pluripotent stem cells by real time PCR analysis revealed significant reduction of lineage markers such as N-cadherin, cytokeratin 18 and prominin 1. In vitro differentiation experiments as well as germline transmission proved full pluripotency of the enriched pluripotent stem cell population. The enrichment of pluripotent cells will be a major advantage in standardizing mESC / miPSC maintenance culture and differentiation experiments as well as generation of transgenic mice.