Abstract text: The plasma membrane plays a central role in the deposition of plant cell walls. Phytosterols constitute approximately one-third of plasma membrane lipids and regulate cellulose synthesis, yet the underlying mechanisms remain unknown. Here, we investigate how the membrane sterol composition impacts cellulose synthesis during secondary cell wall formation. In Arabidopsis roots, sterol perturbation led to defects in protoxylem vessel morphology and SCW patterning, as well as changes in cell wall composition. Our findings indicate that sterols regulate both the spatial organization and biochemical composition of secondary cell walls in planta. To dissect these effects, we introduce several experimental tools. First, we used the dexamethasone-inducible VND7 system in Arabidopsis protoplasts to quantify membrane dynamics during secondary cell wall formation, enabling high-throughput pharmacological screening combined with live-cell imaging. Using this system, we confirmed defects in secondary cell wall deposition upon sterol perturbation. Second, we observed Cellulose Synthase (CESA) and microtubule marker dynamics, together with cellulose reporter probes, to further investigate how sterol perturbation affects SCW deposition. Finally, we present a new protoplast–giant unilamellar vesicle fusion strategy to study plant membrane proteins in defined lipid environments with controlled sterol compositions, enabling direct investigation of how specific sterols modulate CESA complex behavior.