Marek's disease (MD) vaccines reduce the incidence of MD but cannot control virus shedding. To develop new vaccines, it is essential to elucidate mechanisms of immunity to Marek's disease virus (MDV) infection. In this regard, gamma delta (γδ) T cells may play a significant role in prevention of viral spread and tumor surveillance. Here we demonstrated that MDV vaccination induced interferon (IFN)-γ+CD8α+ γδ T cells and transforming growth factor (TGF)-β+ γδ T cells in lungs. γδ T cells from MDV-infected chickens exhibited cytotoxic activity. Importantly, γδ T cells from the vaccinated/challenged group exhibited maximum cytotoxic activity following ex vivo stimulation. These results suggest that MDV vaccines activate effector γδ T cells which may be involved in the development of protective immune responses against MD. Further, it was demonstrated that MDV infection increases the frequency of a subpopulation of γδ T cells expressing membrane-bound TGF-β in MDV-infected birds.