Purpose: To compare the dynamic changes of CD4 CD25 thymocytes development in vivo and in vitro. Methods: Fetal mice thymus lobes were removed at the age of 14.5 days and cultured in complete RPMI-1640 with 20% FBS in vitro. Phenotypes of the thymocytes were analyzed by FACS and the number of cells per lobe was counted. Results: It revealed that from day 14.5 to day 19, the number and the percentage of the CD4 CD25 thymocytes varied similarly as their development as in vitro culture in 6 days. The CD4 CD25/CD4 percentage was 8.19%, 16.41%, 24.06%, 13.62%, 8.10%, 2.35% for each and the CD4 CD25 /CD25 was 0.34%, 1.08%, 2.40%, 10.59%, 32.51%, 34.04% respectively. In vitro data showed that during the 6 days in vitro culture, the CD4 CD25/CD4 percentage was 58.29%, 12.14%, 6.08%, 17.78%, 9.06%, 4.04% and the CD4 CD25/CD25 percentage was 3.75%, 10.81%, 17.20%, 51.93%, 61.64%, 80.06% for each. Conclusions: These results suggested that the in vitro and in vivo CD4 CD25 thymocytes development was similar, with its peak development on the 4 day in vitro and the 17 days in vivo. The above data might help to understand the development of naturally arising CD4 CD25 regulatory T cells.