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Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spect rometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESC) during differentiation induced by bone morphogenetic protein (BMP) and removal of hESC growth factors. Of 5222 proteins identified, 1399 were phosphorylated on 3067 residues. Approximately 50% of these phosphosites were regulated within 1 hr of differentiation induction, revealing a complex interplay of phosphorylation networks spanning different signaling pathways and kinase activities. Among the phosphorylated proteins was the pluripotency-associated protein SOX2, which was SUMOylated as a result of phosphorylation. Using the data to predict kinase-substrate relationships, we reconstructed the hESC kinome; CDK1/2 emerged as central in controlling self- renewal and lineage specification. The findings provide new insights into how hESC exit the pluripotent state and present the hESC (phospho)proteome resource as a complement to existin g pluripotency network databases.