Regulatory T (Treg) cells are a distinct subset of CD4+ T cells. Instead of triggering adaptive immunity, they suppress immune responses. Small numbers of Treg cells reside within lymphoid organs and peripheral tissues, but their contribution to immune tolerance is so significant that defects in Treg cell function cause catastrophic immune disorders. Since they were first discovered 20 years ago, efforts have been made to understand the differences in developmental processes between Treg cells and conventional T cells that determine the ultimate fate of the overall T-cell population. Transcription factor Foxp3 is crucial for Treg cell differentiation, but it is not the whole story. Owing to recent advances in Treg cell research, we are now on the verge of appreciating the comprehensive mechanisms underlying Treg cell generation. Here, we discuss major discoveries, active study topics and remaining questions regarding Treg cell development.