Regulation of integrin activity enables leukocytes to circulate freely, avoiding inappropriate adhesion while maintaining the ability to adhere quickly at sites of infection or inflammation. This regulation involves at least two components: affinity for ligand and affinity-independent avidity effects such as lateral mobility. Using lymphocyte function associated antigen-1 (LFA-1) as a model, we investigated the role of integrin release from cytoskeletal motion constraints in response to the chemokine stromal cell-derived factor-1 (SDF-1α) in this process. All experiments were done in primary T cells to avoid nonphysiological activation processes often seen with the use of cell lines. We found that SDF-1α releases LFA-1 from cytoskeletal constraints as effectively as does cytochalasin D. The resultant increased diffusion is correlated with a robust increase in LFA-1-mediated adhesion under physiological shear stress. We further investigated the role of the highly conserved GFFKR sequence in the LFA-1 cytoplasmic domain. We report that the GFFKR sequence is both necessary and sufficient for regulation of the SDF-1α-triggered proadhesive release from cytoskeleton interactions. While this does not address the role of transient SDF-1α-induced conformational changes in the activation process, these results strongly suggest that any model of chemokineinduced LFA-1 activation must take into account chemokine-induced integrin lateral mobility. In addition, these results have ramifications for models of differential binding of LFA-1 to surface-bound vs. soluble intercellular adhesion molecule-1. © 2012 the American Physiological Society.