Thymocytes mature in a series of stages by migrating through specific areas of the thymus and interacting with other cells to receive the necessary developmental signals; however, little is known about the molecular mechanisms governing this migration.We report that murine thymocytes with a knockout mutation in α-PAK (p21-activated kinase)-interacting exchange factor (PIX; Arhgef6), an activator of Rho GTPases, showed greatly increased motility and altered morphology in two-dimensional migration on ICAM-1. αPIX- was also required for efficient positive selection, but not negative selection, of thymocytes. TCR signaling was normal in αPIX- thymocytes, indicating that the effects of αPIX on positive selection are largely independent of TCR signaling. αPIX2 thymocytes also paused less during migration in the thymic cortex, interacted less with ICAM-1 coated beads, and could overcome TCR stop signals, consistent with defective scanning behavior. These results identify αPIX as a regulator of thymocyte migration and subsequent arrest that is linked to positive selection. [ABSTRACT FROM AUTHOR]