Engagement of very late Ag-4 (integrin α 4 β 1) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin α L β 2). This form of integrin trans-regulation in T cells requires the binding of paxillin to the α 4 integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an α 4 mutation (α 4 (Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an α 4-binding fragment of paxillin that blocks the α 4-paxillin interaction, selectively blocked VCAM-1 stimulation of α L β 2-dependent cell migration. The α 4-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the α 4 tail resulted in much less α 4 β 1-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked α 4 β 1 stimulation of α L β 2-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin α L β 2 by α 4 β 1.