There is an increasing realization that the failure of adoptive therapy with cytotoxic T lymphocytes in the autologous setting, at least in part, results from the lack of help from antigen-specific CD4+ T cells. To incorporate these cells into this treatment strategy, it is not known whether currently used ex vivo culture conditions are adequate for expanding and charting these T cells with the desired qualities for optimal in vivo activity. In this study, we show that stimulation with agonistic antibodies to CD3 plus CD28 (anti-CD3/CD28), a commonly used method for CD4+ T cell expansion, is unable to expand dendritic-cell-activated hepatitis B virus (HBV)-specific CD4+ T cells to clinical relevant numbers. Whereas, in combination with interleukin(IL)-7 and IL-15, it leads to a 4000-fold expansion of HBV-specific CD4+ T cells in 2 weeks. This outcome is correlated with the anti-apoptosis effect of IL-7 and IL-15. Importantly, antigen specificity is preserved during expansion. Although a late addition of IL-2 to the anti-CD3/CD28-expanding cultures also results in robust expansion, this expansion condition renders HBV-specific CD4+ T cells more sensitive to cytokine withdrawal-, activation-, and transforming growth factor-beta-induced cell death compared to those expanded in IL-7 and IL-15. Moreover, NKG2D rather than 4-1BB, whose ligands are constitutively expressed on tumor cells, is significantly up-regulated on IL-7/IL-15-expanded HBV-specific CD4+ T cells, and its engagement promotes expansion and interferon-gamma production by these cells and thus may serve to provide co-stimulation to T cells once they reach tumor tissues. Collectively, these results may have important therapeutic implications for adoptive T cell therapy.