The key feature of T cells is their T cell receptor (TCR), which renders them specific to antigen. Immature double positive (DP) T cells develop in the thymus into mature single positive (SP) cells. During this maturation process, developing T cells are positively selected if their receptors are able to receive a signal from self-antigen and negatively selected by the proapoptotic Bim-mediated mechanism if they bind self-antigen too strongly. Meanwhile, DP cells are lineage selected into either CD4 or CD8 SP cells. Some self-reactive cells are agonist-selected and upregulate Foxp3 to become regulatory T cells (Treg), which suppress immune responses. The temporal order of these selection processes is not well understood due to a lack of tools to investigate temporal changes of T cell development in vivo. To determine the sequence of and relationship between these events, this project used the novel technology Timer of Cell Kinetics and Activity (Tocky), which analyses the maturation of a fluorescent timer (Timer) protein to show temporal changes in transcription. Here, Tocky was developed and used to study in vivo transcriptional dynamics of Foxp3 and a gene immediately downstream TCR signalling, Nr4a3, to analyse Treg differentiation and TCR signalling downstream activities. In Nr4a3-Tocky mice, Timer+ cells were enriched with cells that undergo negative and agonist selection. Nr4a3-Tocky:BimKO/KO mice showed that Bim-mediated negative selection occurred rapidly at both DP and SP stages in the most immature Timer+ cells. Foxp3 was detected after Bim transcription was downregulated in Timer+ cells, following persistent TCR signals. Intriguingly, in Nr4a3-Tocky:BimKO/KO mice Timer+ cells were enriched with cells that escaped negative selection and highly self-reactive Foxp3+ cells were accumulated in both DP and SP fractions. Based on this work, a new model for the temporal order of thymic selection processes and T cell maturation is proposed.