This study investigates phase transitions in the assembly of multivalent signaling proteins, revealing that interactions between synthetic multivalent macromolecules, including multi-domain proteins and RNA, lead to sharp, liquid-liquid demixing phase separations, generating micron-sized liquid droplets in aqueous solutions. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin regulatory protein, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) interacting with its partners Nck and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity toward the actin nucleation factor, Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property can be controlled by kinases. The widespread occurrence of multivalent systems suggests that phase transitions are likely used to spatially organize and biochemically regulate information throughout biology. Covalent and non-covalent interactions between multivalent small molecules are central to polymer chemistry and supramolecular chemistry. These fields have produced theories and experimental demonstrations of sharp transitions between small assemblies and macroscopic polymer gels (sol-gel transitions) as the degree of bonding increases. The transition point depends on physical properties of the monomeric species such as valency and affinity. The polymer can have a variety of physical forms, ranging from phase-separated liquid to crystalline solid. In biology, interactions between multivalent entities are found in many processes, ranging from extracellular carbohydrate-lectin binding to intracellular signaling to RNA metabolism to chromatin organization. Biological multivalency has been studied most extensively in the context of extracellular ligands binding to cell surface receptors, where antibody-receptor and carbohydrate-lectin systems can assemble into crosslinked networks. These networks are typically precipitates, but liquid-like gels have also been described. Multivalency has been less studied in the context of intracellular molecules, which often share characteristics of high valency, modest affinity and long, flexible connections between binding elements. The study shows that interactions between the Src homology 3 (SH3) domain and its proline-rich motif (PRM) ligand produce sharp transitions to polymer, and that the macroscopic properties of the polymer are influenced by the phase transition. The phase separation is driven by the unique ability of multivalent SH3m-PRMn interactions to create large assemblies. The data suggest that the phase transition coincides with a sol-gel transition, and that the droplet phase contains large polymeric species and likely has undergone a sol-gel transition. The behavior observed in vitro is mirrored in cells. Co-expression of mCherry-SH3 and eGFP-PRM5 in HeLa cells results in the formation of cytoplasmic puncta containing both fluorophThis study investigates phase transitions in the assembly of multivalent signaling proteins, revealing that interactions between synthetic multivalent macromolecules, including multi-domain proteins and RNA, lead to sharp, liquid-liquid demixing phase separations, generating micron-sized liquid droplets in aqueous solutions. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin regulatory protein, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) interacting with its partners Nck and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity toward the actin nucleation factor, Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property can be controlled by kinases. The widespread occurrence of multivalent systems suggests that phase transitions are likely used to spatially organize and biochemically regulate information throughout biology. Covalent and non-covalent interactions between multivalent small molecules are central to polymer chemistry and supramolecular chemistry. These fields have produced theories and experimental demonstrations of sharp transitions between small assemblies and macroscopic polymer gels (sol-gel transitions) as the degree of bonding increases. The transition point depends on physical properties of the monomeric species such as valency and affinity. The polymer can have a variety of physical forms, ranging from phase-separated liquid to crystalline solid. In biology, interactions between multivalent entities are found in many processes, ranging from extracellular carbohydrate-lectin binding to intracellular signaling to RNA metabolism to chromatin organization. Biological multivalency has been studied most extensively in the context of extracellular ligands binding to cell surface receptors, where antibody-receptor and carbohydrate-lectin systems can assemble into crosslinked networks. These networks are typically precipitates, but liquid-like gels have also been described. Multivalency has been less studied in the context of intracellular molecules, which often share characteristics of high valency, modest affinity and long, flexible connections between binding elements. The study shows that interactions between the Src homology 3 (SH3) domain and its proline-rich motif (PRM) ligand produce sharp transitions to polymer, and that the macroscopic properties of the polymer are influenced by the phase transition. The phase separation is driven by the unique ability of multivalent SH3m-PRMn interactions to create large assemblies. The data suggest that the phase transition coincides with a sol-gel transition, and that the droplet phase contains large polymeric species and likely has undergone a sol-gel transition. The behavior observed in vitro is mirrored in cells. Co-expression of mCherry-SH3 and eGFP-PRM5 in HeLa cells results in the formation of cytoplasmic puncta containing both fluoroph