The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs. Display omitted •Mice expressing analog-sensitive Cdk1 allow identification of Cdk1 substrates•Many Cdk1 substrates in ESCs are chromatin-bound at poised or transcribed genes•Cdk1 phosphorylates a large number of epigenetic regulators•Cdk1 phosphorylates and inactivates H3K79 methyltransferase Dot1l Cdk1 drives mitotic entry and progression. Michowski et al. generated mice expressing analog-sensitive Cdk1. The authors found that in embryonic stem cells, Cdk1 phosphorylates a large number of epigenetic regulators and controls the global epigenetic landscape. Decreased Cdk1 activity during differentiation allows coordinated expression of differentiation genes.