Th17 and regulatory T (Treg) cells is critical toward revealing pro-inflammatory immune responses, especially in autoimmune diseases. However, there is no standard protocol to monitor the development of these cells over time in vivo. This protocol details a method to generate Th1, Th17, and Treg cells in a T cell-induced colitis model in vivo and monitor their dynamic changes, which can be used for further investigations in their development from naive CD4+ T cells in specific gene knockouts and background strains. This protocol starts with the isolation of naive CD4+ CD45RBhigh T cells from C57BL/6 IL-17Agfp mice followed by their ip injection into recombinase, activating gene-1-deficient (Rag1−/−) mice. At the indicated time points, mice are killed, and mesenteric lymphocytes and murine lamina propria mononuclear cells are isolated and subjected to flow cytometric analysis to assess the development of Th1, Th17, and Treg cells and Th2, Th9, Th22, and follicular T helper (Tfh) cells. This protocol can be completed within 8 weeks. CD4+ T cells play a central role in the function of the immune system, particularly in adaptive immunity. They help the activity of other immune cells by releasing T cell cytokines. CD4+ T cells can be subdivided into lineages based on immunological functions, specific transcription factors, and cytokines: Th1, Th2, Th9, Th17, Th22, Tfh and Treg cells. Aberrant activation of Th1, Th17, Th22 and Tfh cells has been implicated in many autoimmune and inflammatory diseases, whereas excessive Th2 activity causes allergic diseases. Conversely, impaired function of Treg cells causes fatal inflammatory disorders both in human and mouse. 1, 2 Although T helper and Treg cell development has been extensively studied in vitro, 3, 4 their development in vivo is not as well understood. Here, we use the classical T cell transfer model of colitis originally described by Powrie 5 to investigate the developmental characteristics of various T helper and Treg cells in vivo. Initially, Th1 cells were thought to dominate the pathogenesis of colitis. 5 Recent studies indicate that Th17 cells are even more important than Th1 cells in this process. 6 In total, this system could be used as a model to investigate the development of Th1/Th17 and Treg cells in vivo since CD4+ T cells isolated from mesenteric lymph nodes (MLN) and colonic lamina propria (LP) express specific phenotypes and functional characteristics of Th and Treg cells. 7 Using Th17 reporter mice in a colitis model, we herein provide a detailed protocol and define the dynamics of T helper and Treg cell development in vivo.