Gliotoxin is an immunosuppressive mycotoxin long suspected to be a potential virulence factor of Aspergillus fumigatus. Recent studies using mutants lacking gliotoxin production, however, suggested that the mycotoxin is not important for pathogenesis of A. fumigatus in neutropenic mice resulting from treatment with cyclophosphomide and hydrocortisone. In this study, we report on the pathobiological role of gliotoxin in two different mouse strains, 129/Sv and BALB/c, that were immunosuppressed by hydrocortisone alone to avoid neutropenia. These strains of mice were infected using the isogenic set of a wild type strain (B-5233) and its mutant strain (gliPΔ) and the the glip reconstituted strain (gliP_R). The gliP gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The gliPΔ strain was significantly less virulent than strain B-5233 or gliP_R in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233, gliPΔ, and gliP_R strains showed the following: (i) deletion of gliP abrogated gliotoxin production, as determined by high-performance liquid chromatography analysis; (ii) unlike the CFs from strains B-5233 and gliP_R, gliPΔ CFs failed to induce proapoptotic processes in EL4 thymoma cells, as tested by Bak conformational change, mitochondrial-membrane potential disruption, superoxide production, caspase 3 activation, and phosphatidylserine translocation. Furthermore, superoxide production in human neutrophils was strongly inhibited by CFs from strain B-5233 and the gliP_R strain, but not the gliPΔ strain. Our study confirms that gliotoxin is an important virulence determinant of A. fumigatus and that the type of immunosuppression regimen used is important to reveal the pathogenic potential of gliotoxin.