In conclusion, we have demonstrated that NFAT5 is required for the replication of R5-tropic subtype B and subtype C HIV-1 isolates in response to MTb-co-infection of human PBMC and MDM. Functional NF-κB interaction with the viral LTR is also required, but fully intact NF-κB binding elements are unable to compensate for the loss of NFAT5 recruitment to the viral promoter. In addition, we have demonstrated that MTb infection or stimulation with the TLR2 ligand PAM3Cys, induces NFAT5 gene expression in human monocytes. Furthermore, we have shown that MTb stimulation of NFAT5 depends on TLR pathway signaling molecules, including MyD88, IRAK1, and TRAF6. Taken together, the findings presented here enhance the general understanding of the innate immune response to MTb infection by showing that NFAT5 is a major mediator of TLR-dependent gene expression; its importance for gene regulation is likely applicable to other MTb- and TLR-regulated genes. Moreover, these data provide molecular insights into MTb regulation of HIV-1 transcription, thereby elucidating several new targets for therapeutic interventions aimed at controlling TB/HIV-1 co-infection.