As an obligate biotroph, Blumeria graminis f. sp. hordei (Bgh) cannot be grown in an axenic culture, and instead must be cultivated on its host species, Hordeum vulgare (barley). In this study an in vitro system utilizing n-hexacosanal, a constituent of the barley cuticle and known inducer of Bgh germination, was used to cultivate Bgh and differentiate conidia up to the appressorial germ tube stage for analysis. Transcriptomic and proteomic profiling of the appressorial germ tube stage revealed that there was a significant shift towards energy and protein production during the pre-penetrative phase of development, with an up-regulation of enzymes associated with cellular respiration and protein synthesis, modification and transport. Glycosidic linkage analysis of the cell wall polysaccharides demonstrated that during appressorial development an increase in 1,3- and 1,4-linked glucosyl residues and xylosyl residues was detected along with a significant decrease in galactosyl residues. The use of this in vitro cultivation method demonstrates that it is possible to analyse the pre-penetrative processes of Bgh development in the absence of a plant host.