NF-kappaB is a transcriptional regulator involved in many biological processes including proliferation, survival, and differentiation. Recently, we reported that expression and activity of NF-kappaB is comparatively low in undifferentiated human embryonic stem (ES) cells, but increases during differentiation. Here, we found a lower expression of NF-kappaB p65 protein in mouse ES cells when compared with mouse embryonic fibroblast cells. Protein levels of NF-kappaB p65 and relB were clearly enhanced during retinoic acid-induced differentiation. Furthermore, increased DNA binding activity of NF-kappaB in response to TNF-alpha, an agonist of NF-kappaB signaling, was seen in differentiated but not undifferentiated mouse ES cells. Taken together with our previous data in human ES cells, it is likely that NF-kappaB expression and activity of the NF-kappaB signaling pathway is comparatively low in undifferentiated ES cells, but increases during differentiation of ES cells in general.