In this study, we have analyzed the in vivo dynamics of the interaction between polyclonal Foxp3⁺ Treg cells, effector T (Teff) cells, and DCs in order to further our understanding of the mechanisms of Treg cell‐mediated suppression. Cotransfer of polyclonal activated Treg cells into healthy mice attenuated the induction of EAE. Suppression of disease strongly correlated with a reduced number of Teff cells in the spinal cord, but not with Treg cell‐mediated inhibition of Th1/Th17 differentiation. Cotransfer of Treg cells with TCR‐Tg Teff cells followed by immunization by multiple routes resulted in an enhanced number of Teff cells in the lymph nodes draining the site of immunization without an inhibition of Teff‐cell differentiation. Fewer Teff cells could be detected in the blood in the presence of Treg cells and fewer T cells could access a site of antigen exposure in a modified delayed‐type hypersensitivity assay. Teff cells recovered from LNs in the presence of Treg cells expressed decreased levels of CXCR4, syndecan, and the sphingosine phosphate receptor, S1P1 (sphingosine 1‐phosphate receptor 1). Thus, polyclonal Treg cells influence Teff‐cell responses by targeting trafficking pathways, thus allowing immunity to develop in lymphoid organs, but limiting the number of potentially auto‐aggressive cells that are allowed to enter the tissues.