To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2−/− and TLR4−/− animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88−/− mice failed to control acute and chronic M. avium growth and succumbed 9–14 wk postinfection. Infected TLR2−/− mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88−/− animals, while TLR4−/− mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88−/− mice revealed massive destruction of lung tissue not present in WT, TLR2−/−, or TLR4−/− mice. In addition, MyD88−/− and TLR2−/−, but not TLR4−/−, mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88−/− and TLR2−/− macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88−/− mice. Similarly, MyD88−/− mice displayed a profound defect in IFN-γ response that was not evident in TLR2−/− or TLR4−/− mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-γ responses.