Bidirectional signaling of integrin α_IIbβ₃ requires the β₃ cytoplasmic domain. To determine the sequence in the β₃ cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin α_IIb, and mutants of β₃ with truncations at sites COOH terminal to T⁷⁴¹, Y⁷⁴⁷, F⁷⁵⁴, and Y⁷⁵⁹. Truncation at Y⁷⁵⁹ did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of β₃ is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F⁷⁵⁴, Y⁷⁴⁷, or T⁷⁴¹ completely abolished integrin activation. A point mutation replacing Y⁷⁵⁹ with alanine also abolished integrin activation. Thus, the T⁷⁵⁵NITY⁷⁵⁹ sequence of β₃, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y⁷⁵⁹ in a population of β₃ during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.