Updated project metadata. Protein kinase signalling is a major mechanism by which embryonic stem cell pluripotency and differentiation is controlled. However, the pathways and components that regulate embryonic stem cell identity have not been systematically defined. Here, we employ FGF4 signalling as a model system to investigate phosphoproteome dynamics in differentiating mouse embryonic stem cells. We report identification and quantitation of more than 10,000 phosphopeptides, of which hundreds of phosphophoylation sites are regulated more than 2-fold by acute FGF4 stimulation. We hypothesise that phosphorylation sites in this dataset are relevant for regulating the transition of mouse embryonic stem cells from pluripotency towards lineage specific differentiation.