ABSTRACT The adaptive immune system provides remarkable defense against fast-evolving pathogens. In the meantime, tolerance mechanisms coevolved to prevent deleterious immune responses, among which regulatory T (Treg) cells play a dominant, active role. The adaptive immune system is established during postnatal development through continuous differentiation and egress of T and B cells into the peripheral lymphoid and non-lymphoid organs. It appears that the cells of the adaptive immune system that emerge at the early and late stages of life are programmed differently in their immunological function. Which factors are present at particular stages of life to program the function of immune cells differently is an unsolved mystery. How T and B cells developed at different stages of life respond to specific immunological challenges also remains to be fully explored. Revealing these features of the adaptive immune system will offer us better ways to manipulate immune cells for the treatment of immunological diseases. Genetic tracing tools are needed to address these important questions. However, despite significant progress in the field, tracing and investigating T and B cells emerging at given stages of life remain a technical challenge. To solve this issue, we propose a robust, versatile method to trace T and B cells during their early differentiation in experimental mice. In our preliminary experiments, we have successfully engineered this mouse strain. Specifically, we took advantage of transient Rag1 expression during early T and B cell differentiation and used it to drive the expression of three proteins to trace T and B cells with tamoxifen, to report Rag1 expression with fluorescent protein GFP, and to deplete Rag1-expressing cells with diphtheria toxin when necessary. We have validated all these genetic elements and their functions in the mice. In our proposed research, we will improve our methods to meet various experimental requirements. Furthermore, we will use Treg cells as a proof of concept of our technology in revealing the differential immunosuppressive activity and gene regulatory networks of Treg cells developed at the early and later stages of life in mouse models of autoimmune diseases. In summary, our study generates a robust, versatile genetic toolbox in experimental mice to report, trace, and acutely deplete developing T or B cells when necessary at given stages of life, offering a powerful approach to address many significant questions about adaptive immunity. Our research also produces insights into different Treg functions specified during postnatal development and reveals novel factors and mechanisms controlling Treg-mediated immune tolerance.