Pistachio nuts are an economically important commodity produced by many countries. They can be colonized by mycotoxigenic fungi, especially Aspergillus flavus, resulting in contamination with aflatoxins (AFs), especially aflatoxin B 1 (AFB 1), a Class 1a carcinogen. The objectives were to examine the effect of interactions between the two key abiotic factors, temperature and water activity (a w ) on (a) in vitro growth and AFB 1 production by four strains of A. flavus isolated from pistachio nuts, on a milled pistachio nut medium modified ionically (NaCl) and non-ionically (glycerol) in the range 20–35°C and 0.995–0.85 a w , (b) colonization of layers of raw pistachio nuts stored at different interacting temperature x a w conditions and on relative AFB 1 production and (c) develop models to produce contour maps of the optimal and marginal boundary conditions for growth and AFB 1 production by up to 4 strains of this species. On pistachio nut-based media, optimum growth of four strains of A. flavus was at 0.98–0.95 a w and 30–35°C. Optimum AFB 1 production was at 30–35°C and 0.98 a w . No significant differences in growth was found on ionic and non-ionically modified media. Colonization of layers of raw pistachio nuts was slower and contamination with AFB 1 significantly less than in in vitro studies. Contour maps based on the pooled data for up to four strains ( in vitro, in situ) showed the optimum and marginal conditions for growth and AFB 1 production. These data can be used to identify those conditions which represent a high, intermediate or low risk of colonization and AFB 1 contamination in the pistachio nut processing chain. These results are discussed in the context of the development of appropriate intervention strategies to minimize AFB 1 contamination of this economically important commodity.