In S. aureus, the low molecular weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed deletion mutants of bshA (sa1291), which encodes the glycosyl transferase that catalyzes the first step of BSH biosynthesis, and fosB (SA2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. HPLC analysis, which quantified thiol extracts, revealed some variability in the amounts of BSH present across S. aureus strains. Deletion of fosB leads to a decrease in BSH levels. The fosB and bshA mutants of COL and a USA300 isolate, upon further characterization, were found to be sensitive to H2O2 and exhibited decreased NADPH levels vs. the isogenic parents. Microarray analyses of COL and the isogenic bshA mutant revealed increased expression of genes involved in staphyloxanthin synthesis in the bshA mutant relative to COL under thiol stress. However, the bshA mutant of COL demonstrated decreased survival when compared to that of the parent in human whole blood survival assays; likewise, the naturally BSH-deficient strain SH1000 survived less well than its BSH-producing isogenic counterpart. Thus, the survival of S. aureus under oxidative stress is facilitated by BSH, possibly via a FosB-mediated mechanism, independent of its capability to produce staphyloxanthin.