Genetic modification is an important tool in embryonic stem (ES) cell research and requires efficient promoter systems. Here, we have compared the transcriptional activities of three ubiquitous promoters, elongation factor-1alpha (EF1alpha), phosphoglycerate kinase-1 (PGK), and cytomegalovirus (CMV), during propagation and differentiation of mouse (m) ES cells by using stable mES cell lines expressing enhanced green fluorescent protein (EGFP) under each of these promoters. In undifferentiated ES cells, the EGFP expression driven by the EF1alpha was most stable, followed by the PGK, whereas the down-regulation of EGFP expression driven by the CMV promoter was most significant during propagation up to passage 35. A similar pattern for the activities of these promoters was observed in embryoid bodies (EBs) during 14 days of differentiation, with brighter EGFP signals driven by the EF1alpha promoter versus the other two. Moreover, the EF1alpha and PGK promoters, but not CMV, were effective in almost all mES cell-differentiated neuronal cells, cardiomyocytes, and visceral endoderm cells, with the fluorescent signal intensity higher for EF1alpha and even for PGK. The CMV promoter yielded a weak fluorescent signal in about 60-80% of these differentiated cells, while a few differentiated cells with the CMV promoter showed bright EGFP expression like that with the EF1alpha promoter. These results extend previous observations for the activities of these promoters in mES cells and provide new information for choosing appropriate promoters to facilitate genetic modification of mES cells.