House dust mites are cultured to obtain mite allergen material to produce allergen extracts (vaccines) for diagnostic tests, immunotherapy, and research purposes. Research laboratories and manufacturers have their own culturing protocols to grow these mites and these may vary between manufacturers and between research laboratories. The temperature at which mites are cultured may influence the allergen composition, allergen ratio of Der 1: Der 2 and endotoxin levels in the extracts produced from these cultured mites. In order to produce standardized and uniform extracts, across the industry and in various research laboratories, the influence of culture conditions must be understood. Here we determined how temperature affects mite population growth rates, dynamics of allergen production, Der f 1: Der f 2 ratio and endotoxin levels in extracts made from Dermatophagoides farinae mites cultured at 20 and 25 °C. We found that Der f 1 and Der f 2 accumulated exponentially in the cultures with Der f 1 accumulating faster than Der f 2. When the live mite populations peaked, the ratios for Der f 1: Der f 2 were 4.1 and 4.7 for cultures reared at 20 and 25 °C, respectively. Most of the Der f 1 and Der f 2 allergen in whole cultures is not in mite bodies and is lost when the mite material is washed. Thus, if the ratio of Der f 1 and Der f 2 is an important consideration for commercial and research extracts, then the temperature at which the mites are cultured and the collection procedure are important considerations.