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Embryonic stem cell (ESC) identity and self‐renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here, we show that three members of the Ccr4‐Not complex, Cnot1, Cnot2, and Cnot3, play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts, these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs, Cnot1, Cnot2, and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self‐renewal pathways or core transcription factors. Instead, they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly, these Cnot genes are also necessary for the maintenance of human ESCs, and silencing them mainly lead to TE and primitive endoderm differentiation. Together, our results indicate that Cnot1, Cnot2, and Cnot3 represent a novel component of the core self‐renewal and pluripotency circuitry conserved in mouse and human ESCs. S TEM  C ELLS   2012;30:910–922

Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation