T cell development is dependent on the integration of multiple signaling pathways, although few links between signaling cascades and downstream nuclear factors that play a role in thymocyte differentiation have been identified. We show here that expression of the HMG box protein TOX is sufficient to induce changes in coreceptor gene expression associated with β-selection, including CD8 gene demethylation. TOX expression is also sufficient to initiate positive selection to the CD8 lineage in the absence of MHC–TCR interactions. TOX-mediated positive selection is associated with up-regulation of Runx3, implicating CD4 silencing in the process. Interestingly, a strong T cell receptor–mediated signal can modify this cell fate. We further demonstrate that up-regulation of TOX in double positive thymocytes is calcineurin dependent, linking this critical signaling pathway to nuclear changes during positive selection.