Ambient temperature affects the lifespan of cold-blooded organisms such as Drosophila melanogaster. To better understand what influences the lifespan of an adult fruit fly, we tested whether developmental temperature could affect stress responses used as surrogate markers for the aging process. When 2-day-old adult flies developed at two representative temperatures (18°C and 25°C) were challenged with three stresses (starvation, oxidation, and heat), both male and female flies developed at 18°C exhibited stronger resistance to all three stresses compared to those developed at 25°C. Nutrient composition analyses showed that fat, protein, and glycogen levels increased when male flies were developed at 18°C. These differences in stress resistance by developmental temperature were sustained even between 30-day-old male flies of two groups aged at the same temperature. We also showed that development at a lower temperature represented by 18°C significantly downregulates anti-microbial peptide genes, AttA and DptB, of Imd pathway.