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.