Tolerance to self-Ags is generated in the thymus. Both epithelial and hematopoietic thymic stromal cells play an active and essential role in this process. However, the role of each of the various stromal cell types remains unresolved. To our knowledge, we describe the first comparative analysis of several types of thymic hematopoietic stromal cells (THSCs) for their ability to induce CD4 tolerance to self, in parallel with the thymic epithelium. The THSCs--two types of conventional dendritic cells (cDCs), plasmacytoid dendritic cells, macrophages (MΦs), B lymphocytes, and eosinophils--were first characterized and quantified in adult mouse thymus. They were then examined in reaggregated thymic organ cultures containing mixtures of monoclonal and polyclonal thymocytes. This thymocyte mixture allows for the analysis of Ag-specific events while avoiding the extreme skewing frequently seen in purely monoclonal systems. Our data indicate that thymic epithelium alone is capable of promoting self-tolerance by eliminating autoreactive CD4 single-positive thymocytes and by supporting regulatory T cell (Treg) development. We also show that both non-Treg CD4 single-positive thymocytes and Tregs are efficiently deleted by the two populations of cDCs present in the thymus, as well as to a lesser extent by MΦs. Plasmacytoid dendritic cells, B lymphocytes, and eosinophils were not able to do so. Finally, cDCs were also the most efficient THSCs at supporting Treg development in the thymus, suggesting that although they may share some characteristics required for negative selection with MΦs, they do not share those required for the support of Treg development, making cDCs a unique cell subset in the thymus.