Recent reports have provided evidence for cross-talk between regulatory T (Treg) cells and natural killer T (NKT) cells. However, it is unclear whether NKT cells play a role in the differentiation of Treg cells. By employing NKT cell-abundant V alpha 14 TCR transgenic (Tg) and NKT cell-deficient CD1d knock-out (KO) mice, we examined the effects of NICT cells on the in vitro differentiation of induced Treg (iTreg) cells with IL2 and TGF beta. We found that iTreg induction from CD1d KO mice was significantly increased compared to the control. Also, the addition of isolated NKT cells from V alpha 14 TCR Tg mice to naive CD4(+) T cells from CD1d KO mice during iTreg differentiation caused a remarkable reduction of iTreg cells. Through IFN gamma neutralization, we showed that this reduction was mediated by IFN gamma. Furthermore, the main source of IFN gamma during iTreg differentiation was NK1.1(-)CD4(+)Foxp3(-) T cells. This finding implied that early-activated NKT cells induced Th1-type cells and subsequently underwent apoptosis. Taken together, our results suggest that NKT cells inhibit the in vitro development of iTreg cells by increasing IFN gamma. (C) 2011 Elsevier Inc. All rights reserved.