Lipopolysaccharide (LPS) structural modifications have been shown to specifically affect the pathogenesis of many Gram-negative pathogens. In Francisella , modification of the lipid A component of LPS resulted in a molecule with no to low endotoxic activity. The role of the terminal lipid A phosphates in host recognition and pathogenesis was determined using a Francisella novicida mutant that lacked the 4′ phosphatase enzyme (LpxF). The lipid A of this strain retained the phosphate moiety at the 4′ position and the N-linked fatty acid at the 3′ position on the diglucosamine backbone. Studies were undertaken to determine the pathogenesis of this mutant strain via the pulmonary and subcutaneous routes of infection. Mice infected with the lpxF -null F. novicida mutant by either route survived primary infection and subsequently developed protective immunity against a lethal wild-type (WT) F. novicida challenge. To determine the mechanism(s) by which the host controlled primary infection by the lpxF -null mutant, the role of innate immune components, including Toll-like receptor 2 (TLR2), TLR4, caspase-1, MyD88, alpha interferon (IFN-α), and gamma interferon(IFN-γ), was examined using knockout mice. Interestingly, only the IFN-γ knockout mice succumbed to a primary lpxF -null F. novicida mutant infection, highlighting the importance of IFN-γ production. To determine the role of components of the host adaptive immune system that elicit the long-term protective immune response, T- and B-cell deficient RAG1 ^−/− mice were examined. All mice survived primary infection; however, RAG1 ^−/− mice did not survive WT challenge, highlighting a role for T and B cells in the protective immune response.