The complexity of environmental factors is often greatly simplified in ecological studies, which may result in outcomes that do not accurately reflect biological responses under natural circumstances. For example, in many turtle species with temperature-dependent sex determination (TSD), cooler incubation temperatures induce the expression of genes necessary for testis development (i.e. Dmrt1), while warmer temperatures induce the expression of genes necessary for ovary development (i.e. Cyp19A1). However, these patterns were characterized using constant temperatures, which do not accurately reflect the thermal fluctuations experienced during natural incubation conditions. We hypothesized that the timing of upregulation for genes involved in sexual differentiation differs between constant and fluctuating temperatures. We predicted that fluctuating incubation conditions would delay the induction of sexual differentiation because embryos receive thermal cues for both sexes. To investigate this, we exposed red-eared slider (Trachemys scripta) embryos to two conditions that produce all male hatchlings (26 °C and 26 ± 3 °C) and two that produce all female hatchlings (31 °C and 31 ± 3 °C). Gonads were sampled during multiple points across the middle third of development, when sex is sensitive to temperature effects, and the expression of Dmrt1 and Cyp19A1 was quantified. From these data, we will be able to determine how the expression of gonad-specific genes varies between constant and fluctuating temperatures. Designing experiments with high ecological relevancy will prove critical in understanding how biological systems operate outside the laboratory.