The lack of unambiguous Foxp3⁺ Treg cell–specific surface markers has prompted the development of various transgenic mouse lines with Foxp3-dependent reporter activity, which involved different fluorochromes and transgenic strategies, including coexpression of multiple transgenes, such as Cre recombinase. Since then, Foxp3 transcriptional reporter has proven to be an indispensable tool to identify and isolate viable Foxp3⁺ Treg cell populations. However, the physiologic Treg cell pool is functionally heterogeneous and consists of intrathymically (tTreg) and peripherally (pTreg) induced Treg cells, which may confound interpretation of data relying on indiscriminatory Foxp3-fluorochrome reporter expressed in all Treg cells. In this chapter, we describe how the dual Foxp3^RFP/GFP reporter can be exploited to discriminate both developmental sublineages based on tTreg cell lineage–specific GFP/Cre recombinase activity, in conjunction with Foxp3-driven RFP expression in all Foxp3⁺ Treg cells, and provide guidelines for experimental design and implementation. We also elaborate on the possibility to exploit GFP/Cre expression of Foxp3^RFP/GFP reporter mice for the manipulation of gene expression (activation and inactivation), such as lineage tracing and in vivo ablation of tTreg cells, while sparing pTreg cells.