Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4 + T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4 +CD40L + but not CD4 +CD40L − T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4 +CD40L + T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA −CD45RO +CCR7 −CD62L −ICOS −). To determine the specificity of CD4 +CD40L + T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4 +CD40L + and CD4 +CD40L − T cells by flow cytometry. We further demonstrated that sorted CD4 +CD40L +, but not CD4 +CD40L − fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4 + T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.