Phase separation of signaling molecules promotes T cell receptor (TCR) signal transduction. A 12-component TCR signaling pathway was reconstituted on model membranes, starting with TCR activation and ending with actin assembly. When TCR phosphorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling both in vitro and in human Jurkat T cells. These clusters were enriched in kinases but excluded phosphatases, and enhanced actin filament assembly by recruiting and organizing actin regulators. These results demonstrate that protein phase separation can create a distinct physical and biochemical compartment that facilitates signaling. Reconstitution of a T cell signaling pathway and correlative cellular studies reveal how phase separation of molecules into microclusters can promote biochemical reactions and signaling responses. Upon TCR activation, many cell surface receptors and downstream signaling molecules coalesce into micron- or submicron-sized clusters. However, the effect of this clustering on signal transduction is poorly understood. TCR signaling is a well-studied example of this general phenomenon. TCR signaling proceeds through a series of biochemical reactions that can be viewed as connected modules. In the upstream module, the TCR is phosphorylated by Lck, a membrane-bound protein kinase of the Src family. TCR phosphorylation is opposed by a transmembrane phosphatase, CD45. The phosphorylated cytoplasmic domains of the TCR complex recruit and activate the cytosolic tyrosine kinase ZAP70. In the intermediate module, ZAP70 phosphorylates the transmembrane protein LAT on multiple tyrosine residues. These phosphotyrosines are binding sites for adapter proteins Grb2 and Gads, which further interact with Sos1 or SLP-76. Components of the LAT complex activate several downstream modules that mediate calcium mobilization, mitogen-activated protein kinase (MAPK) activation, and actin polymerization. LAT and its binding partners coalesce into micron- or submicron-sized clusters at the plasma membrane upon TCR activation. Elimination of these microclusters by deletion of key components impairs downstream signaling and transcriptional responses. However, effects due to loss of clusters have not been distinguished from those due to loss of component molecules. Nor do we understand the changes in biochemistry and consequent signaling that emerge specifically when signaling molecules are organized from an unclustered to a clustered state. To explore the mechanism of formation and functional consequences of T cell microclusters, we reconstituted a TCR signaling pathway from purified components. We used supported bilayers composed of a defined, simple lipid composition to substitute for the plasma membrane. We initially reconstituted the intermediate module of the TCR signaling cascade, composed of phosphorylated LAT (pLAT), Grb2, and Sos1. Multivalent interactions between these proteins are thought to drive the formation of signaling microclusters on the T cell membrane. We prepared fluorescentPhase separation of signaling molecules promotes T cell receptor (TCR) signal transduction. A 12-component TCR signaling pathway was reconstituted on model membranes, starting with TCR activation and ending with actin assembly. When TCR phosphorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling both in vitro and in human Jurkat T cells. These clusters were enriched in kinases but excluded phosphatases, and enhanced actin filament assembly by recruiting and organizing actin regulators. These results demonstrate that protein phase separation can create a distinct physical and biochemical compartment that facilitates signaling. Reconstitution of a T cell signaling pathway and correlative cellular studies reveal how phase separation of molecules into microclusters can promote biochemical reactions and signaling responses. Upon TCR activation, many cell surface receptors and downstream signaling molecules coalesce into micron- or submicron-sized clusters. However, the effect of this clustering on signal transduction is poorly understood. TCR signaling is a well-studied example of this general phenomenon. TCR signaling proceeds through a series of biochemical reactions that can be viewed as connected modules. In the upstream module, the TCR is phosphorylated by Lck, a membrane-bound protein kinase of the Src family. TCR phosphorylation is opposed by a transmembrane phosphatase, CD45. The phosphorylated cytoplasmic domains of the TCR complex recruit and activate the cytosolic tyrosine kinase ZAP70. In the intermediate module, ZAP70 phosphorylates the transmembrane protein LAT on multiple tyrosine residues. These phosphotyrosines are binding sites for adapter proteins Grb2 and Gads, which further interact with Sos1 or SLP-76. Components of the LAT complex activate several downstream modules that mediate calcium mobilization, mitogen-activated protein kinase (MAPK) activation, and actin polymerization. LAT and its binding partners coalesce into micron- or submicron-sized clusters at the plasma membrane upon TCR activation. Elimination of these microclusters by deletion of key components impairs downstream signaling and transcriptional responses. However, effects due to loss of clusters have not been distinguished from those due to loss of component molecules. Nor do we understand the changes in biochemistry and consequent signaling that emerge specifically when signaling molecules are organized from an unclustered to a clustered state. To explore the mechanism of formation and functional consequences of T cell microclusters, we reconstituted a TCR signaling pathway from purified components. We used supported bilayers composed of a defined, simple lipid composition to substitute for the plasma membrane. We initially reconstituted the intermediate module of the TCR signaling cascade, composed of phosphorylated LAT (pLAT), Grb2, and Sos1. Multivalent interactions between these proteins are thought to drive the formation of signaling microclusters on the T cell membrane. We prepared fluorescent