The trimeric Cdk7/cyclin H/Mat1 complex functions in cell cycle regulation, as the Cdk-activating kinase (CAK), and in transcription, as a module of the general transcription factor TFIIH. As a component of TFIIH, Cdk7 phosphorylates serine-5 and serine-7 of the carboxy-terminal domain (CTD) of RNA polymerase II and can also directly phosphorylate transcription factors to regulate gene expression. Here we have investigated the function of the Cdk7/cyclin H/Mat1 complex in murine embryonic stem (ES) cells and preimplantation embryos to determine whether it regulates the unique cell cycle structure and transcriptional network of pluripotent cells. We demonstrate that depletion of cyclin H leads to differentiation of ES cells independent of changes in cell cycle progression. In contrast, we observed that developmental genes are acutely upregulated after cyclin H downregulation, likely perturbing normal ES self-renewal pathways. Consistent with its function in ES cells, cyclin H depletion from mouse embryos also leads to defects in the expansion of the inner cell mass (ICM) of blastocysts, a transient pluripotent stem cell population in vivo. Thus, cyclin H has an essential function in promoting the self-renewal of pluripotent stem cells from blastocyst-stage embryos. Collectively, these studies demonstrate a critical and novel role for cyclin H in maintaining ES cell identity and suggest that cyclin H has important functions in early embryonic development.