Francisella tularensis (Ft) is the causative agent of tularemia. Due to its aerosolizable nature, high virulence, and low infectious dose, Ft poses a significant bioterror threat. It is classified as a category A select agent by the CDC and is a priority for therapeutics and vaccine development. Towards this goal, a greater understanding of its pathogenic mechanisms was investigated. The Ft genome is predicted to encode 31 Major Facilitator Superfamily (MFS) transporters, and the nine-member Ft phagosomal transporter (Fpt) subfamily possesses homology to virulence factors in other intracellular pathogens such as Legionella pneumophila. Intracellular survival and replication is critical to Ft pathogenesis. The central hypothesis tested was that these Fpt transporters are involved in normal intracellular survival and replication of Ft and host response to infection and will serve as viable targets for attenuation and vaccine development. Mutational analysis identified three fpt mutants (LVSδfptB, LVSδfptE, and LVSδfptG) that exhibited altered intracellular replication kinetics and attenuation of virulence in mice compared to wild-type Ft LVS. Vaccination of mice with a single dose of each mutant strain was protective against lethal intraperitoneal challenge. Like Ft LVS, these mutant strains colonized the lungs, liver and spleen of infected mice. However, there was often a delay in colonization and these strains did not replicate in the organs to the same extent as Ft LVS. The fpt mutants were completely cleared from the organs within 3-4 weeks after infection. While the cytokine responses to infection with these mutant strains were largely unaffected in murine macrophages when compared to Ft LVS, there were pronounced differences in cytokine profiles in the livers of infected mice. The altered in vivo cytokine responses may contribute to the attenuation observed in these strains. Future work will investigate the function of each of the transporters and the cellular and molecular mechanisms that underlie the attenuation of these strains. Overall, the data presented herein confirms the hypothesis that members of the Fpt family are critical factors in the pathogenesis of Ft and represent promising targets for development of efficacious live-attenuated vaccines.