Cow's milk allergy in children is often of short duration, which makes this disorder an interesting clinical model for studies of tolerance to dietary antigens. Here, we studied T cell responses in 21 initially allergic children who, after a milk-free period of >2 mo, had cow's milk reintroduced to their diet. Children who outgrew their allergy (tolerant children) had higher frequencies of circulating CD4⁺CD25⁺ T cells and decreased in vitro proliferative responses to bovine β-lactoglobulin in peripheral blood mononuclear cells (PBMCs) compared with children who maintained clinically active allergy. No significant difference in proliferative activity stimulated by the polyclonal mitogen phytohemagglutinin was observed between the two groups. Depletion of CD25⁺ cells from PBMCs of tolerant children led to a fivefold increase in in vitro proliferation against β-lactoglobulin. This suggests that tolerance is associated with the appearance of circulating CD4⁺CD25⁺ regulatory T (Treg) cells that are capable of suppressing the effector T cells generated 1 wk after reintroduction of cow's milk. The suppressive function of the CD4⁺CD25⁺ Treg cells was shown to be partly cell contact dependent. Collectively, our study provides human data to suggest that mucosal induction of tolerance against dietary antigens is associated with the development of CD4⁺CD25⁺ Treg cells.