Updated project metadata. Understanding the mechanisms of pluripotency maintenance and exit are important for developmental biology and regenerative medicine. However, studies of pluripotency and post-translational modifications of proteins are scarce. To systematically profile protein crotonylation in mouse pluripotent stem cells (PSCs) in different culture conditions, we used affinity purification of crotonylated peptides, TMT labeling, and LC-MS/MS. Our study included PSCs in ground, metastable, and primed state, as well as metastable PSCs undergoing early pluripotency exit. We have successfully identified 8,102 crotonylation sites in 2,578 proteins, among which 3,628 sites in 1,426 proteins were with high-confidence. These high-confidence crotonylated proteins are enriched for factors involved in functions related to pluripotency such as RNA biogenesis, central carbon metabolism, and proteasome function. Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of crotonylation in other cell fate transitions.