The granulomatous response is a characteristic histological feature of Mycobacterium tuberculosis infection responsible for organism containment. The development of cell-mediated immunity is essential for protection against disease, as well as being required for maintenance of the sequestering granulomatous response. Trehalose 6,6′-dimycolate (TDM; cord factor), a glycolipid associated with the cell wall of mycobacteria, is implicated as a key immunogenic component in M. tuberculosis infection. Models of TDM-induced hypersensitive granulomatous response have similar pathologies to that of active tuberculosis infection. Prior immunization (sensitization) of mice with TDM results in exacerbated histological damage, inflammation and lymphocytic infiltration upon subsequent TDM challenge. Adoptive transfer experiments were performed to ascertain the cell phenotype governing this response; CD4+ cells were identified as critical for development of related pathology. Mice receiving CD4+ cells from donor TDM-immunized mice demonstrated significantly increased production of Th1-type cytokines IFN-γ and IL-12 within the lung upon subsequent TDM challenge. Control groups receiving naïve CD4+ cells, or CD8+ or CD19+ cells isolated from TDM-immunized donors, did not exhibit an exacerbated response. The identified CD4+ cells isolated from TDM-immunized mice produced significant amounts of IFN-γ and IL-2 when exposed to TDM-pulsed macrophages in vitro. These experiments provide further evidence for involvement of a cell-mediated response in TDM-induced granuloma formation, which mimics pathological damage elicited during M. tuberculosis infection.