Integrin-mediated adhesion of cells to extracellular matrix proteins triggers a variety of intracellular signaling pathways including a cascade of tyrosine phosphorylations. In many cell types, the cytoplasmic focal adhesion tyrosine kinase, FAK, appears to be the initial protein that becomes tyrosine-phosphorylated in response to adhesion; however, the molecular mechanisms regulating integrin-triggered FAK phosphorylation are not understood. Previous studies have shown that the integrin ␤ 1 , ␤ 3 , and ␤ 5 subunit cytoplasmic domains all contain sufficient information to trigger FAK phosphorylation when expressed in single-subunit chimeric receptors connected to an extracellular reporter. In the present study, ␤ 3 cytoplasmic domain deletion and substitution mutants were constructed to identify amino acids within the integrin ␤ 3 cytoplasmic domain that regulate its ability to trigger FAK phosphorylation. Cells transiently expressing chimeric receptors containing these mutant cytoplasmic domains were magnetically sorted and assayed for the tyrosine phosphorylation of FAK. Analysis of these mutants indicated that structural information in both the membrane-proximal and C-terminal segments of the ␤ 3 cytoplasmic domain is important for triggering FAK phosphorylation. In the C-terminal segment of the ␤ 3 cytoplasmic domain, the highly conserved NPXY motif was found to be required for the ␤ 3 cytoplasmic domain to trigger FAK phosphorylation. However, the putative FAK binding domain within the N-terminal segment of the ␤ 3 cytoplasmic domain was found to be neither required nor sufficient for this signaling event. We also demonstrate that the serine 752 to proline mutation, known to cause a variant of Glanzmann's thrombasthenia, inhibits the ability of the ␤ 3 cytoplasmic domain to signal FAK phosphorylation, suggesting that a single mutation in the ␤ 3 cytoplasmic domain can inhibit both "inside-out" and "outside-in" integrin signaling.