Integrin adhesion receptors can signal in two directions: first, they can regulate cellular behaviors by modulating cellular signaling enzymes ("outside-in signaling"); second, cells can regulate the affinity of integrins ("inside-out signaling") by such pathways. Integrin beta cytoplasmic domains (tails) mediate both types of signaling, and Src family kinases (SFKs) and talin, which bind to beta tails, are important for integrin signaling. Here, we utilized "homology scanning" mutagenesis to identify beta tail mutants selectively defective in c-Src binding and found that amino acid exchanges affecting a combination of an Arg and Thr residue in the integrin beta3 tail control the binding specificity for SFKs but have no effect on talin binding. Using beta tail mutants at these residues, we found that SFK binding to integrin beta tails is dispensable for inside-out signaling but is obligatory for cell spreading, a marker of outside-in signaling. Conversely, we found that point mutations that disrupt talin binding abolish integrin activation, but they do not inhibit SFK binding to the beta3 tail or the initiation of outside-in signaling once the integrins are in a high affinity form. Thus, we show that inside-out and outside-in integrin signaling are mediated by distinct and separable interactions of the integrin beta tails. Furthermore, based on our results, it is possible to discern the relative contributions of the direction of integrin signaling on biological functions in cell culture and, ultimately, in vivo.