Pluripotent stem cells, self-renew indefinitely and possess characteristic protein-protein networks that remodel all through differentiation. How this Y-27632 DOCA happens is poorly understood. Employing quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during differentiation induced by bone morphogenetic protein (BMP) and elimination of hESC development components. Of 5222 proteins recognized, 1399 were phosphorylated on 3067 residues. Roughly 50% of these phosphosites had been regulated inside one hrHER2 of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase routines. Between the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated due to phosphorylation. Utilizing the data to predict kinase-substrate relationships, we reconstructed the hESC kinome, CDK1/2 emerged as central in controlling se renewal and lineage specification. The findings supply new insights into how hESCs exit the pluripotent state and present the hESC (phospho)proteome resource like a complement to existing pluripotency network databases.