Abstract: The dynamics and essential pathways necessary in maintaining robust patterning of the Drosophila embryo are uncovered using a microfluidic platform that selectively controls the temperature of each half of the embryo. Biochemical networks function within the context of their environment, and can be understood and controlled by perturbing their environment in space and time. This approach was applied to investigate the robustness of the antero-posterior patterning network in the Drosophila embryo at the level of maternal, gap and pair-rule gene expression. A microfluidic platform was developed that created a temperature step across a live Drosophila embryo. The temperature and flow profile in the microfluidic channel was characterized both experimentally and by numerical simulation, and the temperature profile within the embryo was predicted by numerical simulation. The microfluidic platform was coupled to confocal microscopy to facilitate capturing real-time dynamics of nuclear movement and formation of protein gradients. Results presented here uncovered new concepts and pathways critical in maintaining robustness. First, the real-time formation of the maternal Bicoid protein gradient in embryos exposed to the temperature step showed that active transport must be considered in forming gradients essential to patterning. Second, a time window critical in maintaining robust Hunchback patterning was identified. This time window was considerably early in development. Third, the RNAi pathway, previously thought to be non-essential to development, was shown to be essential in maintaining robust Even-skipped patterning under environmental perturbations. These three new findings advance current knowledge on mechanisms of robustness and could lend information on general mechanisms that maintain robust cellular functions.