The genetic program of embryonic stem (ES) cells is orchestrated by a core of transcription factors that has OCT4, SOX2, and NANOG as master regulators. Protein levels of these core factors are tightly controlled by autoregulatory and feed-forward transcriptional mechanisms in order to prevent early differentiation. Recent studies have shown that knockdown of Esrrb (estrogen-related-receptor beta), a member of the nuclear orphan receptor family, induces differentiation of mouse ES cells cultured in the presence of leukemia inhibitory factor. It was however not known how knocking down Esrrb exerts this effect. Herein we have identified two ESRRB binding sites in the proximal 5'-untranslated region of the mouse Oct4 gene, one of which is in close proximity to a NANOG binding site. Both ESRRB and NANOG are necessary for maintaining the activity of this promoter in ES cell lines. We have also demonstrated that the two transcription factors interact through their DNA binding domains. This interaction reciprocally modulates their transcriptional activities and may be important to fine-tune ES cell pluripotency. Supporting all of these data, stable transfection of Esrrb in ES cell lines proved sufficient to sustain their characteristics in the absence of leukemia-inhibitory factor. In summary, our experiments help to understand how Esrrb coordinates with Nanog and Oct4 to activate the internal machinery of ES cells.