<p>Gnotobiotic mouse models occupy a unique position in allergy research as the only systems that allow causal links between defined microbial inputs and specific immune outcomes. This review summarises the long-term contributions of the Laboratory of Gnotobiology at the Institute of Microbiology of the Czech Academy of Sciences in Nový Hrádek, covering two decades of mechanistic research following the germ-free (GF) models pioneered by Professor Jaroslav Šterzl. The review is structured around three themes. First, GF BALB/c mice retain full capacity for Th2 sensitisation and mucosal tolerance, but allergy outcomes are strongly influenced by endotoxin content of the mouse chow, an often overlooked variable that should be explicitly controlled. Second, defined microbial colonisation during the perinatal period reduces allergic sensitisation through mechanisms including mucosal tolerance induction, tolerogenic dendritic cell programming, gut barrier restoration, and regulatory immune responses. Third, the microbiota is required not only for sensitisation but also for development of the intestinal mast cell effector compartment that mediates IgE-dependent disease; this function is not restored by a single probiotic strain and likely requires greater microbial complexity. Together, these findings define microbiota–allergy interactions in mechanistic terms. They identify dietary endotoxin as a key experimental confound and establish the perinatal period (encompassing maternal and neonatal microbial exposure) as critical for both prevention of sensitisation and maturation of effector responses. They also show that single-strain probiotics and complex microbiota reconstitution target distinct components of allergic disease. The review concludes by highlighting methodological considerations, open questions, and future directions, including postbiotics and extracellular vesicle-based approaches in allergy modulation.</p>