Stable heritable restriction of the ubiquitous powdery mildew disease is a desirable trait for agri- and horticulture. In barley (Hordeum vulgare), loss-of-function mutant alleles of the Mildew resistance locus o (Mlo) gene confer broad-spectrum resistance to almost all known isolates of the fungal barley powdery mildew pathogen, Blumeria graminis f.sp. hordei. Despite extensive cultivation of barley mlo genotypes, mlo resistance has been durable in the field. Mlo genes are present as small families in the genomes of all higher plant species. The presumed negative regulatory role of particular members in plant immunity is evolutionarily conserved, as powdery mildew resistant mlo mutants have also been described in Arabidopsis thaliana, tomato (Solanum lycopersicum) and pea (Pisum sativum). Barley Mlo encodes a plasma membrane-localized seven-transmembrane domain protein of unknown biochemical activity. Here, we review the known requirements for mlo-mediated disease resistance in barley and Arabidopsis and reflect current views regarding Mlo function. We discuss additional mlo mutant phenotypes recently discovered in Arabidopsis and present a meta-analysis of the phylogenetic relationships within the Mlo family. Finally, we consider the novel versatile tools for functional analysis and targeted genome modification that can be used to induce mlo-based powdery mildew resistance in virtually any plant species.