Zymoseptoria tritici is a fungal phytopathogen and the causative agent of Septoria Tritici blotch (STB), one of the most economically and agriculturally significant foliar diseases of wheat. Infection causes the formation of necrotic lesions on leaf tissue, leading to a decrease in tissue available for photosynthesis, significantly reducing crop yield. Fludioxonil is a phenylpyrrole fungicide which hyperactivates the HOG pathway, an essential stress activated signalling pathway which regulates cellular responses to a wide range of environmental stresses and is essential for virulence. We have identified several constituents of a two-component signalling pathway, a His-Asp phosphorelay system, which regulate HOG activation in response to fludioxonil. We have found that ZtNik1, a Group III hybrid histidine kinase, and the downstream phosphotransferase response regulator, ZtSsk1, are essential for fludioxonil sensitivity inZ. tritici. Loss of either of these proteins, or deletion of ZtHog1, the stress activated MAP kinase, leads to full resistance to the fungicide. Deletion of ZtAft1, a bZIP transcription factor, also confers partial fludioxonil resistance suggesting that it functions as a downstream target of ZtHog1. In addition to fludioxonil, ZtSsk1 is required for the activation of ZtHog1 in response to a variety of stresses, including osmotic and oxidative stress, suggesting that it is a master regulator of HOG signalling. Furthermore, we have also found evidence suggesting that ZtNik1 plays both positive and negative roles in the control of ZtHog1 phosphorylation.