Salmonella infections are a major threat to global health and consist primarily of acute gastroenteritis and enteric fever. Although gastroenteritis is more common, enteric fever caused by Salmonella Typhi and Paratyphi is responsible for a disproportionate share of human morbidity and mortality. A distinctive characteristic of enteric fever is a prolonged incubation period and illness. Mechanisms of enteric fever pathogenesis are incompletely understood due to the human host-specificity of S. Typhi and Paratyphi. However, we have developed a humanized mouse model of enteric fever using NOD-scid-IL2rγnull mice engrafted with human hematopoietic stem cells, which recapitulates many of the pathological features of human typhoid. The ability of S. Typhi and S. Paratyphi to cause progressive disease in this model indicates that human macrophages are required for virulence. Analysis of high-density transposon libraries has revealed fundamental differences in the virulence determinants of S. Typhi and nontyphoidal Salmonella. S. Typhi virulence in humanized mice requires metabolic genes, Vi-capsule, LPS O-antigen and iron acquisition systems, but not the classical Salmonella virulence factors PhoP and Salmonella Pathogenicity Island-2, nor the typhoid toxin. Moreover, S. Typhi and Paratyphi A cause persistent infection of cultured human macrophages, in contrast to S. Typhimurium, which rapidly induces apoptosis. Salmonella-induced macrophage death is dependent on SPI-2 and specific SPI-2 effectors that are present in S. Typhimurium but absent from enteric fever serovars, which promote cell death and inhibit NF-κB-dependent cell survival pathways. These observations provide a striking example of the evolution of bacterial virulence by gene loss and suggest that targeting NF-κB might be useful for the treatment of chronic infections caused by intracellular pathogens.