Bacillus cereus is a food-borne pathogen that can produce cereulide in the growth period, which causes food poisoning symptoms. Due to its resistance to heat, extreme pH, and proteolytic enzymes, cereulide poses a serious threat to food safety. Temperature, pH, and a_w can influence cereulide production, but there is still a lack of research with multi-environmental impacts. In this study, the effects of temperature (15~45 °C), pH (5~8), and a_w (0.945~0.996) on the emetic reference strain B. cereus F4810/72 growth, cereulide production, relevant ces genes (cesA, cesB, cesP), and transcription regulators genes (codY and abrB) expression at transcription level were studied. B. cereus survived for 4~53 h or grew to 6.85~8.15 log₁₀ CFU/mL in environmental combinations. Cereulide accumulation was higher in mid-temperature, acidic, or high a_w environments. Increased temperature resulted in a lower cereulide concentration at pH 8 or aw of 0.970. The lowest cereulide concentration was found at pH 6.5 with an increased a_w from 0.970 to 0.996. Water activity had a strong effect on transcriptional regulator genes as well as the cesB gene, and temperature was the main effect factor of cesP gene expression. Moreover, environmental factors also impact cereulide synthesis at transcriptional levels thereby altering the cereulide concentrations. The interaction of environmental factors may result in the survival of B. cereus without growth for a period. Gene expression is affected by environmental factors, and temperature and pH may be the main factors influencing the correlation between B. cereus growth and cereulide formation. This study contributed to an initial understanding of the intrinsic link between the impact of environmental factors and cereulide formation and provided valuable information for clarifying the mechanism of cereulide synthesis in combined environmental conditions.