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Francisella tularensis is the etiological agent of tularemia, a severe and potentially fatal disease in humans. It is extremely infectious by the aerosol route, being thought to cause disease in humans with an infectious dose as small as one to ten organisms, which led to its weaponization by several nations and classification as a category A select agent by the Centers for Disease Control and Prevention. An intracellular pathogen, relatively little is known about the mechanisms by which Francisella is capable of successfully modulating host cell processes to escape its phagosome and replicate within the cytosol and what genes beyond the Francisella pathogenicity island are required. Furthermore, in the context of aerosol exposure, it is unknown what cells F. tularensis initially interacts with and the overall contribution of those interactions to inhalational tularemia. I initiated this study by generating an in vitro model system to study interactions of F. tularensis with epithelial cell lines in tissue culture. Utilizing this system, I determined that F. tularensis LVS was capable of adherence to human epithelial cell lines of alveolar (A549), bronchial airway (HBE), and cervical carcinoma (HEp-2) origin. Furthermore, LVS was capable of invading these cell lines and growing productively within them. In order to detect genes important for virulence in this system, I generated a ~15,000 member transposon library in virulent strain Schu S4 that was could be screened in a high-throughput manner by transposon site hybridization. As uptake in the in vitro epithelial cell line system was relatively inefficient, I screened this library through human primary macrophages. Results of the screen implicated 207 genes as negatively selected in the human macrophage model. Of these, I generated mutants in genes residing in a locus of the Francisella chromosome, FTT1236, FTT1237, and FTT1238, to determine their virulence phenotypes. Mutants in these genes demonstrated significant vulnerability to complement-mediated lysis as compared with wild type Schu S4. Analysis of purified LPS and capsule from these mutants further showed that they had marked defects in O-antigen and capsular polysaccharide biosynthesis. Complementation of these mutants restored surface polysaccharide biosynthesis and further determined that FTT1236 and FTT1237 compose an operon, as a mutation in FTT1236 is polar onto FTT1237. Characterization of the intracellular defect of these mutants in the absence of active complement demonstrated that they were taken up more efficiently by primary human macrophages than wild type Schu S4 and were capable of phagosomal escape but exhibited reduced intracellular growth. Microscopic analysis of macrophages infected with mutant bacteria revealed that, as early as 16 hpi, these macrophages exhibited signs of cell death. In contrast, cells infected with Schu S4 exhibited a healthy, spread morphology as late as 32 h, despite significantly more extensive F. tularensis cytosolic replication. Quantitation of cell death by the release of lactate dehydrogenase, signifying membrane permeability, confirmed that mutants in FTT1236, FTT1237, and FTT1238 induced early cell death in infected macrophages as compared with wild type Schu S4. Together, this work contributes to our understanding of the factors, such as O-antigen and capsule, required for and genes involved in Francisella's lifecycle as an intracellular pathogen.