Technical Abstract: Robust microorganisms are needed to consume both hexose and pentose sugars and to withstand, survive, and function in the presence of stress factors common to fermentations of lignocellulose hydrolysates, including the inhibitors furfural, 5-hydroxymethylfurfural (HMF), and ethanol. Pichia stipitis NRRL Y-7124 is one of the natural yeast strains best able to utilize biomass because it is able to ferment hexoses and the pentose, xylose, to ethanol. Previous studies have shown that mineral and nitrogen source composition supplied to fermentations significantly impact the ability of P. stipitis to efficiently convert high xylose concentrations to economically recoverable ethanol. Current studies focused on the dependency of inhibitor tolerance on culture nutrition and physiological state. Resistance of cells to ethanol, furfural, and HMF was generally greater in stationary-phase cells than log-phase cells, despite the greater exposure of stationary cells to ethanol. Consistent with this, the specific productivity of detoxification products (furfuryl alcohol or furan-2,5-dimethanol) from spikes of furfural or HMF, respectively, increased as cultures progressed into stationary phase. However, when xylose was substrate, ethanol resistance was greater for log-phase cells than stationary cells. Amino acid enrichment of the growth medium significantly enhanced ethanol tolerance if xylose was the carbon source, but had no impact if glucose was. Regardless of the carbon source, amino acid enrichment of the culture medium enhanced the ability of cells to resist furfural and HMF exposure. Mineral compositions had little impact on inhibitor resistance except stationary-phase xylose-grown cells were more susceptible to inhibitor exposure when magnesium sulfate level was too high. These findings shape process strategies to produce a tolerant yeast population, and then to foster and sustain tolerance during growth and ethanol fermentation.