Despite extensive research on regulatory T cells (Tregs) since their rebirth more than twenty years ago, the cellular and molecular mechanisms by which they act to suppress immune responses remain largely elusive. In vitro suppression assays are instrumental in the functional identification of these cells. However, suppressive mechanisms defined in in vitro assays might not be relevant to situations in vivo. Advances in live tissue and intravital imaging technologies combined with the ability to grow large numbers of Tregs for in vivo experimentation have created an opportunity to analyze Treg function in vivo in their native environment in real-time. Two-photon laser-scanning microscopic studies of Treg control of lymph node priming suggest that Tregs exert their function by limiting T helper (Th) cell access to dendritic cells (DCs). In the absence of Tregs, Th cells initially form transient interactions with DCs that lead to arrest of the Th cells and to the formation of stable conjugates between Th cells and DCs. In the presence of increasing number of Tregs, Th cell arrest and their prolonged contacts with DCs are progressively inhibited. The reduced DC contacts in the presence of Tregs are associated with suppressed proliferation and differentiation of Th cells. Expansion of such analysis to peripheral tissues together with the development of functional reporter mice will help to further elucidate the mode of operation of Tregs in vivo.