CD8(+) cytotoxic and CD4(+) helper/inducer T cells develop from common thymocyte precursors that express both CD4 and CD8 molecules. Upon T cell receptor signaling, these cells initiate a differentiation program that includes complex changes in CD4 and CD8 expression, allowing identification of transitional intermediates in this developmental pathway. Little is known about regulation of these early transitions or their specific importance to CD4 and CD8 T cell development. Here, we show a severe block at the CD4(lo)CD8(lo) transitional stage of positive selection caused by loss of the nuclear HMG box protein TOX. As a result, CD4 lineage T cells, including regulatory T and CD1d-dependent natural killer T cells, fail to develop. In contrast, functional CD8(+) T cells develop in TOX-deficient mice. Our data suggest that TOX-dependent transition to the CD4(+)CD8(lo) stage is required for continued development of class II major histocompatibility complex-specific T cells, regardless of ultimate lineage fate.