AbstractLeukocyte integrins of the β2 family are essential for immune cell-cell adhesion. In activated cells, β2 integrins are phosphorylated on the cytoplasmic Thr758, leading to 14-3-3 protein recruitment to the β2 integrin. The mutation of this phosphorylation site impairs cell adhesion, actin reorganization, and cell spreading. Thr758 is contained in a Thr triplet of β2 that also mediates binding to filamin. Here, we investigated the binding of filamin, talin, and 14-3-3 proteins to phosphorylated and unphosphorylated β2 integrins by biochemical methods and x-ray crystallography. 14-3-3 proteins bound only to the phosphorylated integrin cytoplasmic peptide, with a high affinity (Kd, 261 nM), whereas filamin bound only the unphosphorylated integrin cytoplasmic peptide (Kd, 0.5 mM). Phosphorylation did not regulate talin binding to β2 directly, but 14-3-3 was able to outcompete talin for the binding to phosphorylated β2 integrin. X-ray crystallographic data clearly explained how phosphorylation eliminated filamin binding and induced 14-3-3 protein binding. Filamin knockdown in T cells led to an increase in stimulated cell adhesion to ICAM-1–coated surfaces. Our results suggest that the phosphorylation of β2 integrins on Thr758 acts as a molecular switch to inhibit filamin binding and allow 14-3-3 protein binding to the integrin cytoplasmic domain, thereby modulating T-cell adhesion.