Several human diseases are thought to evolve due to a combination of host genetic mutations and environmental factors that include alterations in intestinal microbiota composition termed dysbiosis. Although in some cases, host genetics may shape the gut microbiota and enable it to provoke disease, experimentally disentangling cause and consequence in such host-microbe interactions requires strict control over non-genetic confounding factors. Mouse genetic studies previously proposed Nlrp6/ASC inflammasomes as innate immunity regulators of the intestinal ecosystem. In contrast, using littermate-controlled experimental setups, we recently showed that Nlrp6/ASC inflammasomes do not alter the gut microbiota composition. Our analyses indicated that maternal inheritance and long-term separate housing are non-genetic confounders that preclude the use of non-littermate mice when analyzing host genetic effects on intestinal ecology. Here, we summarize and discuss our gut microbiota analyses in inflammasome-deficient mice for illustrating the importance of littermate experimental design in studying host-microbiota interactions.