Adoptive cell therapies (ACTs) using tumor-reactive T cells have shown clinical benefit and potential for cancer treatment. While the majority of the current ACT are focused on using CD8(+) cytotoxic T lymphocytes (CTL), others have shown that the presence of tumour-reactive CD4(+) T helper (Th) cells can greatly enhance the anti-tumour activity of CD8(+) CTL. However, difficulties in obtaining adequate numbers of CD4(+) Th cells through in vitro expansion can limit the application of CD4 Th cells in ACT. This study optimised the culture conditions for mouse CD4 T cells to provide basic information for animal studies of ACT using CD4 T cells. Taking advantage of the antigen-specificity of CD4(+) Th cells from OT-II transgenic mice, we examined different methodologies for generating antigen-specific CD4(+) Th1 cells in vitro. We found that cells grown in complete advanced-DMEM/F12 medium supplemented with low-dose IL-2 and IL-7 induced substantial cell expansion. These Th cells were Th1-like, as they expressed multiple Th1-cytokines and exhibited antigen-specific cytotoxicity. In addition co-transfer of these CD4(+) Th1-like cells with CD8(+) CTL significantly enhanced tumour regression, leading to complete cure in 80% of mice bearing established B16-OVA. These observations indicate that the CD4(+) Th1-like cells generated using the method we optimized are functionally active to eliminate their target cells, and can also assist CD8(+) CTL to enhance tumor regression. The findings of this study provide valuable data for further research into in vitro expansion of CD4(+) Th1-like cells, with potential applications to cancer treatment involving ACT.