The generation of multiprotein complexes at receptors and adapter proteins is crucial for theactivation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysicalmethods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteinsas they interact with the adapter protein linker for activation of T-cells (LAT) to form multiproteincomplexes. We observed that the binding specificity of these proteins for various LAT tyrosines appearsto be constrained both by the affinity of binding and by cooperative protein-protein interactions. Thesestudies provide quantitative information on how different binding parameters can determine in vivo bindingsite specificity observed for multiprotein signaling complexes.