For the adoptive immunotherapy and the study of cytotoxic T lymphocytes (CTLs) in human, efficient in vitro generation of CTLs is needed. However, it is still difficult to induce T cells specific for naïve antigens in vitro even though dendritic cells (DCs) as potent APCs are used. In this study, we investigated quantitative and qualitative effects of CD4+ T cells during in vitro stimulation of CD8+ T cells from healthy donors using DCs transduced with adenovirus vector expressing human survivin (Adv-survivin). CTLs were not efficiently induced in the absence of CD4+ T cells or in CD25+ depleted CD4+ T cells. When the ratio of CD4+:CD8+ T cells was quantitatively decreased from 2:1 to 1:2, proliferation of CTLs specific for survivin was gradually increased. Because DCs pulsed with HCMV pp65 protein could activate CD4+ T cells to secrete Th1 cytokines, the use of pp65 protein as an adjuvant induced higher numbers and frequencies of CTLs. Furthermore, Th1 conditioning of CD4+ T cells augmented this generation of CTLs. These results suggest that both quantitative and qualitative modulation of CD4+ T cells including the number and Th1 polarization may be required for the efficient induction of CTLs specific for tumor antigens in vitro.