We previously described a fibronectin/VLA-5-dependent impairment of NOD thymocyte migration, correlated with partial thymocyte arrest within thymic perivascular spaces. Yet, NOD thymocytes still emigrate, suggesting the involvement of other cell migration-related alterations. In this context, the aim of this work was to study the role of extracellular matrix ligands, alone or in combination with the chemokine CXCL12, in NOD thymocyte migration. Intrathymic contents of CXCL12, fibronectin, and laminin were evaluated by immunohistochemistry while the expression of corresponding receptors was ascertained by flow cytometry. Thymocyte migration was measured using Transwell chambers and transendothelial migration was evaluated in the same system, but using an endothelial cell monolayer within the chambers. NOD thymocytes express much lower VLA-5 than C57BL/6 thymocytes. This defect was particularly severe in CD4 thymocytes expressing Foxp3, thus in keeping with the arrest of Foxp3 cells within the NOD giant perivascular spaces. We observed an enhancement in CXCL12, laminin, and fibronectin deposition and colocalization in the NOD thymus. Furthermore, we detected altered expression of the CXCL12 receptor CXCR4 and the laminin receptor VLA-6, as well as enhanced migratory capacity of NOD thymocytes toward these molecules, combined or alone. Moreover, transendothelial migration of NOD thymocytes was diminished in the presence of exogenous fibronectin. Our data unravel the existence of multiple cell migration-related abnormalities in NOD thymocytes, comprising both down- and up-regulation of specific responses. Although remaining to be experimentally demonstrated, these events may have consequences on the appearance of autoimmunity in NOD mice.