CD4Foxp3 regulatory T cells (Tregs) are a distinct subset of CD4 T cells that play indispensable role in the maintenance of immune homeostasis and prevention of deleterious immune responses to self-antigens. Tumor necrosis factor (TNF) is a key cytokine in the autoimmune inflammatory responses. The effect of TNF on Treg activity was extensively studied in the past decade. We for the first time reported that TNF through TNFR2 preferentially activates and expands Tregs. Our discovery is increasingly supported by the research community; however, some controversial results were reported. The differential results are likely caused by different experimental condition. A standard experiment protocol can help researchers to obtain more consistent results. In this chapter, we detail methods used to examine in vitro effect of exogenous TNF on the proliferative expansion of Tregs in unfractionated mouse CD4 T cells. The related technic issues are analyzed and discussed.