We previously reported that transcripts encoding the homeoprotein EGAM1N are expressed in preimplantation mouse embryos and embryonic stem (ES) cells, and the exogenous expression of EGAM1N inhibits the differentiation of ES cells. In order to clarify the relationship between the inhibition of differentiation and EGAM1N, we generated mouse MG1.19 ES cells stably expressing EGAM1N. Control transfectants with an empty vector formed relatively flattened cell colonies similar to those observed in parental MG1.19 cells. In contrast, Egam1n transfectants formed tightly aggregated cell colonies with increased localization of CDH1 at cell-to-cell interfaces. The protein levels of pluripotency factors, including TBX3 and SOX2, were also increased. The expression of Tbx3 transcripts was induced, although the level of Sox2 transcripts was almost unchanged. The expression of EGAM1N resulted in no obvious changes in the expression of genes encoding receptors, protein kinases, transcription factors, and their encoded proteins involved in the LIF-STAT3 signaling pathway. Alkaline phosphatase activity, a marker for the undifferentiated state, in Egam1n transfectants was exhibited in a clonal proliferation assay. When differentiation of Egam1n transfectants was induced, progression was prevented with increases in transcript levels of Pou5f1, Sox2, Nanog, K1f4, Tbx3, and their encoded proteins. However, Egam1n transfectants formed relatively flattened-cell layers as observed in the control, indicating that the expression of EGAM1N could not maintain LIF-independent self-renewal of ES cells. Overall, we suggest that expression of EGAM1N could inhibit differentiation, at least in part, by elevating the protein levels of pluripotency factors in MG1.19 ES cells. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.