Embryonic stem (ES) cells have therapeutic potential in regenerative medicine, although the molecular mechanism controlling their pluripotency is not completely understood. Depending on interaction partners most proteins can be involved in several different cellular mechanisms. We screened for novel protein-protein interactions using in situ proximity ligation assays together with specific antibodies directed against known important ES cell proteins. We found that all three core transcription factors, namely Oct4, Sox2 and Nanog, individually formed complexes with nucleophosmin (Npm1). We showed that the Npm1/Sox2 complex was sustained when cells were induced to differentiate by retinoic acid, while decreased in the other differentiation pathways. Moreover, Oct4 also formed individual complexes with translationally controlled tumor protein (Tpt1). Downregulation of Npm1 or Tpt1 increased mRNA levels for genes involved in mesoderm and ectoderm differentiation pathways, respectively, indicative of their involvement in ES cell maintenance. We have here described four novel protein-protein interactions in ES cell involving all three core transcription factors. Our findings improve the current knowledge about ES cell-specific protein networks and indicate the importance of Npm1 and Tpt1 to maintain the ES cell phenotype.