Regulatory T (Treg) cells represent a major roadblock to the induction of antitumor immunity through vaccine approaches. TGF-beta is a cytokine implicated in the generation and maintenance of Treg cells, as well as in their suppressive function. These experiments examined whether the generation of tumor-sensitized Treg cells was TGF-beta dependent and evaluated whether TGF-beta produced by Treg cells blocked the priming of tumor-specific T cells in vaccinated reconstituted lymphopenic mice. We show that tumor-sensitized Treg cells (CD25(+)/FoxP3(+)) obtained from tumor-bearing mice block the generation of tumor-specific T cells in reconstituted lymphopenic mice. Strikingly, this suppression is absent if tumor-sensitized Treg cells are acquired from tumor-bearing mice expressing the dominant-negative TGFbetaRII in T cells. This loss of suppression was a result of the crucial role of TGF-beta in generating tumor-sensitized Treg cells, and not due to the insensitivity of naive or tumor-primed effector T cells to the direct suppressive influence of TGF-beta. We conclude that blocking TGF-beta in a tumor-bearing host can inhibit the induction of highly suppressive tumor-sensitized Treg cells. These data suggest that an integrative strategy combining "up-front" Treg cell ablation followed by vaccination and TGF-beta blockade may limit generation of new tumor-sensitized Treg cells and improve the generation of therapeutic immune responses in patients with cancer.