[Objective] The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-g expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-g expression in different subtypes of T lymphocytes in EIAV-infected horses. [Methods] Peripheral blood mononuclear cells (PBMC) were prepared from horses inoculated with EIAV vaccine stain FDDV (fetal donkey dermal-attenuated virus vaccine), virulent strain LV or saline (health control), were stimulated in vitro with FDDV or PMA + Inomycin. Brefeldin A was added into the cell culture to block protein secretions. Stimulated cells were then labeled with monoclonal antibodies specific for equine CD4 and CD8 surface markers, as well as an IFN-g-specific monoclonal antibody. Flow cytometry was applied to detect CD4+ and CD8+ lymphocytes expressing IFN-g. [Results] IFN-g positive cell population isolated from FDDV-immunized horses was 1.7±0.9% in CD4+ T cells and 6.1±1.2% in CD8+ T cells (n=4). In contrast, only 0.6±0.1% of IFN-g positive cells in CD4+ subset and 2.4±0.9% of IFN-g positive cells in CD8+ subset of T cells were detected for PBMC isolated from LV-infected horses (n=4). [Conclusion] The multi-fluorescence cell flowmetry detecting both the IFN-g expressing cells and subsets of T lymphocytes simultaneously, is specific, stable and reproducible in evaluating antigen-specific response of IFN-g- producing lymphocytes. This is an essential approach to study the protective immunity induced by EIAV vaccine.