Fluconazole (FLC), a triazole with limited activity against Aspergillus species, is frequently used as prophylaxis in leukemia patients and bone marrow transplant recipients. Prior FLC use has been associated with an increasing incidence of invasive aspergillosis in these patients. We hypothesized that prior exposure of Aspergillus fumigatus to FLC could result in altered in vitro susceptibility of this fungus to other, more active triazoles. Thus, we performed serial passages of conidia of 10 clinical isolates of A. fumigatus (all itraconazole [ITC] susceptible) on FLC-containing yeast agar glucose plates. The MICs and minimal fungicidal concentrations (MFCs) of amphotericin B, FLC, ITC, and voriconazole (VRC) for A. fumigatus conidia were measured following four passages on FLC-containing medium according to the National Committee for Clinical Laboratory Standards microdilution method. Serial passages on FLC-containing plates resulted in a fourfold increase in the MFCs (but not the MICs) of ITC for nine isolates. The attenuated ITC fungicidal activity against A. fumigatus following FLC preexposure was medium independent and was also observed against FLC-preexposed A. fumigatus hyphae with the viability staining FUN-1 dye. Moreover, FLC preexposure of A. fumigatus conidia resulted in an analogous increase in the MFCs (but not the MICs) of VRC. Our findings suggest that preexposure of A. fumigatus to FLC attenuates the in vitro fungicidal activity of subsequent ITC use against it. This phenotypic adaptation is not captured by a routine MIC determination but requires MFC measurement. The in vivo significance of this in vitro phenomenon requires further investigation.