The ability of thymocytes to express cytokine genes changes during the different stages of thymic development. Although CD4⁻ CD8⁻ thymocytes are able to produce a wide spectrum of cytokines in response to a T-cell receptor (TcR)-independent stimulus, as they approach the double-positive (DP) CD4⁺ CD8⁺ stage, they lose the ability to produce cytokine. After the DP stage, thymocytes become single-positive CD4⁺ or CD8⁺ thymocytes which reacquire the ability to secrete cytokines. In an attempt to understand the molecular basis of this specific regulation, we use AP-1-luciferase and newly generated NFAT-luciferase transgenic mice to analyze the transcriptional and DNA-binding activities of these two transcription factors that are involved in the regulation of cytokine gene expression. Here, we show that both AP-1 and NFAT transcriptional activities are not inducible in the majority of DP cells but that during the differentiation of DP cells to the mature single-positive stage, thymocytes regain this inducibility. Subpopulation analysis demonstrates that this inducibility is reacquired at the DP stage before the down-modulation of one of the coreceptors. Indeed, AP-1 inducibility, just like the ability to express the interleukin-2 gene, is reacquired during the differentiation of DP TcR^low CD69^low heat-stable antigen (HSA)^high thymocytes to DP TcR^high CD69^high HSA^high cells, which is considered to be the consequence of the first signal that initiates positive selection. We therefore propose that the inability of DP thymocytes to induce AP-1 and NFAT activities is one of the causes for the lack of cytokine gene expression at this stage and that this inducibility is reacquired at the latest stage of DP differentiation as a consequence of positive selection. This could be a mechanism to prevent the activation of DP thymocytes before selection has taken place.