Regulatory Foxp3(+) T cells (T(R)) are indispensable for preventing autoimmune pathology in multiple organs and tissues. During thymic differentiation T cell receptor (TCR)-ligand interactions within a certain increased affinity range, in conjunction with gammac-containing cytokine receptor signals, induce Foxp3 expression and thereby commit developing thymocytes to the T(R) lineage. The contribution of distinct MHC class II-expressing accessory cell types to the differentiation process of Foxp3(+) thymocytes remains controversial, because a unique role in this process has been ascribed to either thymic dendritic cells (tDC) or to medullary thymic epithelial cells (mTEC). Furthermore, it was suggested that the thymic medulla, where the bulk of the negative selection of self-reactive thymocytes takes place, provides a specialized microenvironment supporting T(R) differentiation. Here, we report that the cortex, as defined by cortical thymic epithelial cells (cTEC), is sufficient for supporting T(R) differentiation. MHC class II expression restricted to both cTEC and mTEC or to cTEC alone did not significantly affect the numbers of Foxp3(+) thymocytes. Furthermore, genetic or pharmacologic blockade of thymocyte migration resulted in a prominent accumulation of Foxp3(+) thymocytes in the cortex, demonstrating that secondary signals required for Foxp3 up-regulation exist in the cortex. Our results suggest that mTEC or tDC do not serve as a cell type singularly responsible for T(R) differentiation and that neither the cortex nor the medulla exclusively provides an environment suitable for Foxp3 induction. Instead, multiple accessory cell types probably contribute to the thymic generation of regulatory Foxp3(+) T cells.