The interaction of integrin α 4 β 1 with endothelial VCAM-1 controls the trafficking of lymphocytes from blood into peripheral tissues. Cells actively regulate the affinity of α 4 β 1 for VCAM-1 (activation). To investigate the biological function of α 4 β 1 activation, we isolated Jurkat T cell lines with defective α 4 β 1 activation. Using these cells, we found that α 4 β 1-stimulated α L β 2-dependent cell migration was dramatically reduced in cells with defects in α 4 β 1 activation. These cells required 20 times more VCAM-1 to promote α L β 2-dependent cell migration. This defect was at the level of α 4 β 1 affinity as an activating α 4 β 1 Ab rescued α 4 β 1-stimulated α L β 2-dependent migration. In contrast, migration of α 4 β 1 activation-defective cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus, α 4 β 1 activation determines a set point or threshold at which VCAM-1 can regulate α L β 2-dependent as well as α 4 β 1-dependent cell migration. Changes in this set point may specify preferred anatomical sites of integrin-dependent leukocyte emigration from the bloodstream.