The study investigates the transcriptional regulation of *Nanog* by OCT4 and SOX2, two essential transcription factors for maintaining the pluripotent state of embryonic stem cells (ESCs). The researchers identified a composite sox-oct cis-regulatory element within the *Nanog* proximal promoter that is crucial for its pluripotent-specific expression. This element is conserved over 250 million years of evolution in eutherian mammals. The *Nanog* promoter-EGFP reporter transgene recapitulates endogenous *Nanog* mRNA expression in undifferentiated ESCs and their derivatives. Mutagenesis and *in vitro* binding assays confirmed that OCT4 and SOX2 interact with the *Nanog* promoter. Electrophoretic mobility shift assays showed that the Sox2-Oct4 heterodimer forms more efficiently on the *Nanog* composite element compared to a similar element in *Fgf4*. Chromatin immunoprecipitation experiments demonstrated that OCT4 and SOX2 bind to the *Nanog* promoter in living mouse and human ESCs. RNA interference knockdown of OCT4 and SOX2 reduced *Nanog* promoter activity, establishing a genetic link between these factors and *Nanog* expression. These findings extend the understanding of the pluripotent genetic regulatory network, placing the Sox2-Oct4 complex at the top of the regulatory hierarchy.The study investigates the transcriptional regulation of *Nanog* by OCT4 and SOX2, two essential transcription factors for maintaining the pluripotent state of embryonic stem cells (ESCs). The researchers identified a composite sox-oct cis-regulatory element within the *Nanog* proximal promoter that is crucial for its pluripotent-specific expression. This element is conserved over 250 million years of evolution in eutherian mammals. The *Nanog* promoter-EGFP reporter transgene recapitulates endogenous *Nanog* mRNA expression in undifferentiated ESCs and their derivatives. Mutagenesis and *in vitro* binding assays confirmed that OCT4 and SOX2 interact with the *Nanog* promoter. Electrophoretic mobility shift assays showed that the Sox2-Oct4 heterodimer forms more efficiently on the *Nanog* composite element compared to a similar element in *Fgf4*. Chromatin immunoprecipitation experiments demonstrated that OCT4 and SOX2 bind to the *Nanog* promoter in living mouse and human ESCs. RNA interference knockdown of OCT4 and SOX2 reduced *Nanog* promoter activity, establishing a genetic link between these factors and *Nanog* expression. These findings extend the understanding of the pluripotent genetic regulatory network, placing the Sox2-Oct4 complex at the top of the regulatory hierarchy.