CD19(+)CD5(+) regulatory B cells produce transforming growth factor β (TGF-β) in both mouse and human B-cell leukemias. In this study, TGF-β was uniquely produced by normal human regulatory B cells. TGF-β-producing regulatory B-cell (Br3) responses were characterized through allergic responses to cow's milk. In total, 10 subjects allergic to milk and 13 milk-tolerant subjects were selected following double-blinded, placebo-controlled food challenges. Their peripheral blood mononuclear cells were stimulated in vitro with casein. Following allergen stimulation, the percentage of Br3s among CD5(+) B cells decreased from 11.5% ± 13.7% to 8.0% ± 9.6% (P = 0.042, n = 5) in the milk-allergy group and increased from 14.7% ± 15.6% to 18.9% ± 20.1% (P = 0.006, n = 7) in the milk-tolerant group. However, the numbers of Br3s increased only in the milk-tolerant group, from 1,954 ± 1,058 to 4,548 ± 1,846 per well (P = 0.026), whereas the numbers of Br3s in the milk-allergy group were unchanged [2,596 ± 823 to 2,777 ± 802 per well (P = 0.734)]. The numbers of apoptotic events were similar to the numbers of total Br3 responses. The percentage of non-TGF-β-producing CD5(+) B cells with apoptotic changes increased from 13.4% ± 17.1% to 16.4% ± 20.3% (P = 0.047, n = 5) in the milk-allergy group and remained unchanged [from 9.9% ± 11.9% to 9.3% ± 11.4% (P = 0.099, n = 7)] in the milk-tolerant group. Using carboxyfluorescein succinimidyl ester labeling, we observed that the percentage of proliferating Br3s among CD5(+) B cells was unchanged [from 6.1% ± 2.8% to 6.4% ± 2.9% (P = 0.145)] in the milk-allergy group and increased from 6.8% ± 3.9% to 10.2% ± 5.3% (P = 0.024) in the milk-tolerant group. In conclusion, Br3s proliferated in response to allergen stimulation in the milk-tolerant group and not in the milk-allergy group. TGF-β-producing regulatory B cells (Br3) may be involved in allergy tolerance by negatively regulating the immune system with TGF-β, and this negative regulation may be controlled by apoptosis.