Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote survival in human blood and ultimately facilitate metastases is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Compared to culture supernatants from a wild-type USA300 strain (LAC), those derived from an isogenic hlgABC-deletion strain (LACΔhlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Moreover, LACΔhlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LACΔhlgABC strains caused virtually identical abscesses in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed functional redundancy among two-component leukotoxins in vitro. These findings, along with a requirement of specific growth conditions for leukotoxin expression, may explain the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence.