Phosphorylation is a key post-translational modification involved in many cellular processes. Embryonic stem cells (ESCs), characterized by their self-renewal capacity and pluripotent differentiation potential, are widely used in studies of developmental biology and regenerative medicine. However, existing phosphoproteomic data for ESCs remain limited in throughput, restricting our understanding of phosphorylation-mediated regulatory mechanisms. In this study, we performed high-throughput phosphoproteomic profiling and identified 3,711 phosphoproteins and 11,410 phosphosites.