Recessive mutations in the mildew locus O ( MLO ) gene were first identified as key factors conferring broad-spectrum resistance to powdery mildew in barley. This discovery inspired extensive research on MLOs and novel breeding strategies for powdery mildew resistance by targeting MLO genes in various crops. Over the past two decades, studies have revealed broader roles for MLOs beyond powdery mildew susceptibility, including regulating interactions with diverse pathogens and symbionts, root thigmomorphogenesis, and reproductive development. Recent findings identify MLOs as calcium channels, offering a unifying molecular framework for understanding their diverse biological functions. However, significant challenges remain in comprehensively understanding the cellular and molecular mechanisms underlying MLO functions. In this review, we examine the MLO-related literature to delineate the multifaceted roles of MLOs in plant immunity and development. By integrating published phylogenetic, genetic, biochemical, and molecular studies with original in silico analyses, we propose mechanistic models to contextualize the diverse functions of MLOs with a focus on plant immunity and susceptibility.