T cell development in the thymus requires the establishment of stable interactions with cell-selecting elements such as the cortical epithelium followed by a regulated movement of selected progenitors to the medulla. Cell adhesion and migration are mediated by integrins in a number of biological systems though little is known regarding their function in the thymus. We demonstrated previously that immature CD3loCD69lo double positive human thymocytes adhere avidly to FN via the integrin, VLA4. We now demonstrate that the interaction of mature CD3hiCD69hi thymic subsets with FN triggers migration rather than firm adhesion. Migration requires the engagement of VLA4 in cooperation with VLA5 and both receptors regulate the persistence and directionality of movement. While migration capability is linked to maturation state, ligand concentration determines the efficiency of migration. In fact, FN and the alternatively spliced CS1 site are predominant in the thymic medulla, suggesting an instructive role of this ECM protein in vivo. Our studies identify a novel VLA4 and VLA5/FN-mediated pathway likely to be involved in regulating cell traffic between the cortex and medulla of the thymus. Moreover, the data provides evidence that VLA4 exists in at least two functional states at distinct stages of T cell development. While different states of VLA4 activation have been described on cell lines, this represents the first evidence supporting a biological significance for this integrin property.