Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass 
of blastocysts. Self-renewal of mouse ES cells depends on activation of Stat3 by 
leukaemia inhibitory factor (LIF) in collaboration with bone morphogenetic 
protein signalling. The transcription factor Nanog is essential in maintaining 
pluripotency but the mechanisms involved are poorly understood. Here we examine 
the functional interactions of Nanog with the Stat3 and NFkappaB pathways. Nanog 
and Stat3 were found to bind to and synergistically activate Stat3-dependent 
promoters. We also found that Nanog binds to NFkappaB proteins; however, Nanog 
binding inhibited transcriptional activity of NFkappaB proteins. Endogenous 
NFkappaB activity and target-gene expression increased during differentiation of 
ES cells. Overexpression of NFkappaB proteins promoted differentiation, whereas 
inhibition of NFkappaB signalling, either by genetic ablation of the Ikbkg gene 
or overexpression of the IkappaBalpha super-repressor, increased expression of 
pluripotency markers. We conclude that Nanog represses the pro-differentiation 
activities of NFkappaB and cooperates with Stat3 to maintain pluripotency.