Discovery of a new vaccine is one of the key components to eliminate tuberculosis (TB) disease in the world. Mycobacterium bovis bacille Calmette-Guérin (BCG) is the only licensed TB vaccine, but it has a wide range efficacy and waning immune response. Currently, mucosal immune response and resident-memory T cells in the lungs are proposed as long-lived T cells that improve protection against pulmonary TB infection. Influenza A virus is recognized as a strong inducer of memory T cells in the respiratory tract. We investigated the immunogenicity of sequential immunization with recombinant Influenza A viruses (rIAVs) expressing M. tuberculosis CD4+ T cells epitopes in mice. Intranasal immunization with rIAV PR8 (H1N1) expressing M. tuberculosis Ag85B240-254 epitope (PR8-p25), and rIAV X31 (H3N2) expressing the same epitope (X31-p25) were subsequently given in 6 week-intervals, and the immunogenicity was assessed by IFN-γ ELISpot and intracellular staining flow cytometry. The sequential rIAVs immunization boosted Ag85B-specific IFN-γ producing T cells in the lungs and spleen. A marked proportion of polyfunctional and double IFN-γ and TNF producer CD4+ T cells, was induced in the lungs post immunization with rIAVs compared to each viruses alone (p<0.001). In summary, our influenza-vectored TB vaccines are immunogenic in the lungs and sequential infection with the two different strains of rIAVs can boost the response to the M. tuberculosis Ag85B epitope. This results suggest the possible use of such vectors as tuberculosis vaccines, either alone or to boost the effect of BCG.