Background: γδT cells contribute to immune defense against infectious organisms and some malignancies, but the process of activation and proliferation of these cells is not well understood. It is known that the immune response of γδT cells is not MHC-dependent, but is likely based on direct recognition of surface peptides and non-peptide ligands. This study examined whether DCs and CD4+ T cells can participate in the activation of γδT cells. Method: Peripheral blood γδT cells were co-cultured with CD34-derived autologous DCs and CD4+ T cells using contact-dependent cultures and transwell systems. Proliferation, immunophenotyping, and cytotoxicity assays determined the extent of γδT cell proliferation and cytotoxicity. Results: Human γδT cells expanded 221.3 ± 76-fold in cultures with DCs, and 165.7 ± 76.6-fold with CD4+ T-cells alone. Proliferation was enhanced (1949.8 ± 261.3-fold) when γδT cells were cultured with both DC and CD4+ T cells. Proliferation was contact-dependent, and resulted in the expansion of Vδ1+ or Vδ2+ cells cytotoxic against serveral leukemic cell-lines, but not against allogeneic PHA-induced lymphoid blasts. Ligation of the T-cell receptor with anti-pan-δ Ab significantly up-regulated cytotoxicity against K562, KBM-5 and KG1a, and normal BM, but not against Molt-4, allogeneic EBV-transfected B cells and allogeneic PHA-blasts. Minimal cytotoxic activity was shown against allogeneic marrow colony-forming units granulocyte-macrophage and erythrocyte colony-forming units. Conclusion: DCs can participate in the activation of γδT cells against specific autologous targets, and cytotoxicity can be enhanced by further stimulation via the T-cell receptor.