Conidial germination and differentiation, the so-called prepenetration processes, of the barley powdery mildew fungus (Blumeria graminis f.sp. hordei) are triggered in vitro by very-long-chain aldehydes, minor constituents of barley leaf wax. However, until now it has not been demonstrated that these cuticle-derived molecules also play a significant role in the initiation and promotion of the fungal prepenetration processes in vivo, on the surface of a living plant leaf. In the maize (Zea mays) wax mutant glossy11, which is completely devoid of cuticular very-long-chain aldehydes, germination and appressorial differentiation of B. graminis were strongly impeded. Spraying the mutant leaf surface with aldehyde-containing wild-type wax or pure n-hexacosanal (C26-aldehyde) fully restored fungal prepenetration, whereas maize wild-type leaf surfaces coated with n-docosanoic acid exhibited reduced conidial germination rates of 23%, and only 5% of the conidia differentiated infection structures. In vitro studies were performed to further corroborate the extensive prevention of fungal germination and differentiation in response to artificial surfaces coated with aldehyde-deficient maize wax. Because of its phenotype affecting the B. graminis prepenetration processes, the glossy11 mutation of maize may become a valuable molecular target and genetic tool that could provide a means of developing basal powdery mildew resistance in the globally important crops wheat and barley.