The objectives of this study were to obtain scientific data on the impact that interactions between water stress (water activity (a_w); 0.97, 0.95, 0.92), temperature (34, 37 °C) and CO₂ exposure (350, 650, 1000 ppm) may have on the growth, gene expression of biosynthetic genes (aflD, aflR), and phenotypic aflatoxin B₁ (AFB₁) production by a type strain of Aspergillus flavus on a conducive medium. The study showed that while aw affected growth there was no statistically significant effect of temperature or CO₂ exposure. The effect of these interacting factors on aflD and aflR gene expression showed that at 34 °C there was maximum relative expression of aflD under the control conditions (34 °C, 350 ppm) with a decrease in expression with elevated CO₂ and water stress. For aflR expression at 34 °C, there was a significant increase in expression, but only at 0.92 a_w and 650 ppm CO₂. However, at 37 °C, there was a significant increase in expression of both aflD and aflR at 0.95 and 0.92 a_w and 650 and 1000 ppm CO₂. There was an associated increase in AFB₁ in these treatments. In contrast, at 34 °C there were no significant differences for interacting treatments. This is the first study to examine these three-way interacting climatic factors on growth and mycotoxin production by a strain of A. flavus. This provides data that are necessary to help predict the real impacts of climate change on mycotoxigenic fungi.