In yeasts, cell surface stresses are detected by a family of plasma membrane sensors. Among these, Wsc1 contains an extracellular cysteine-rich domain (CRD), which mediates sensor clustering and is believed to anchor the sensor in the cell wall. Although the formation of Wsc1 clusters and their interaction with the intracellular pathway components are important for proper stress signaling, the molecular mechanisms underlying clustering remain poorly understood. Here, we used the combination of single-molecule atomic force microscopy (AFM) with genetic manipulations to demonstrate that Wsc1 clustering involves disulfide bridges of the CRD. Using AFM tips carrying nitrilotriacetate groups, we mapped the distribution of individual His-tagged sensors on living yeast cells. While Wsc1 formed nanoscale clusters on native cells, clustering was no longer observed after treatment with the reducing agent dithiothreitol (DTT), indicating that intra- or intermolecular disulfide bridges are required for clustering. Moreover, DTT treatment resulted in a significant increase in cell surface roughness, suggesting that disulfide bridges between other cell-wall proteins are crucial for proper cell surface topology. The remarkable sensor properties unravelled here may well apply to other sensors and receptors with cysteine-rich domains throughout biology. Our combined method of AFM with genetic manipulations offers great prospects to explore the mechanisms underlying the clustering of cell surface proteins.