Thymocytes that bind strongly to self-antigens are prevented from becoming naive T cells by several mechanisms. They undergo clonal deletion at two stages of development; wave 1 in immature thymocytes lacking the medulla-homing chemokine receptor, CCR7, or wave 2 in more mature CCR7+ thymocytes. Alternatively, self-reactive thymocytes up-regulate Foxp3 to become T-regulatory cells. Here we describe the differential timing of the two waves of deletion and Foxp3 up-regulation relative to the immature proliferating stage. Proliferating thymocytes were pulse-labelled in normal C57BL/6 mice with 5-ethynyl-2 -deoxyuridine (EdU). Thymocytes progressed into wave 1 (CCR7-) and wave 2 (CCR7+) of clonal deletion around 2 and 5 days after proliferation, respectively. Foxp3 up-regulation occurred between 4 and 8 days after proliferation, predominantly in thymocytes with a Helios+ CCR7+ phenotype. These findings establish a timeline that suggests wave 1 of clonal deletion occurs in the thymic cortex, whereas wave 2 and Foxp3 up-regulation both occur in the thymic medulla.Immunology and Cell Biology accepted article preview online, 29 October 2015. doi:10.1038/icb.2015.95.