Colonic goblet cells are known to produce and secrete mucins and have also been shown to sample and deliver luminal antigens to antigen-presenting cells in the colon lamina propria via the formation of goblet cell associated antigen passages. Gobet cells mediated antigen sampling support the induction and maintenance of oral tolerance to the luminal content. We have previously shown that approximately 10% of goblet cells sample antigens at steady state, primarily in the lower half of the colonic crypts. Whether regulation of colonic immunity is restricted to antigen sampling by goblet cells, or if goblet cells have other features that promote tolerance is currently unknown. To address this question, we quantified goblet cell - immune cell interactions along the crypt – surface axis using confocal microscopy. Our results demonstrate that interactions were regulated by acetyl choline acting on muscarinic receptrors, and by the micrbiota. Our observations show that at steady state, 20% of goblet cells interact with an antigen presenting cells, and that the occurrence is higher in the surface epithelium as compared to the crypts. The acetyl choline agonist carbachol significantly increased interactions in the upper crypt region and surface epithelium. Suppression of the microbiota by broad spectrum antibiotics reduced interactions in the same regions suggesting that the microbiota stimualted these interactions. Taken together, these results show that goblet cell - immune cells interactions are not restricted to goblet cells that sample antigens, as interactions are observed more frequently as compared to antigen sampling. In order to explore the mechanisms that regulate these interaction and identify the sub groups of immune cells that interact with goblet cells we have performed a spatial transcriptomics experiment exploring these interactions in the adult colon at steady state and following antibiotics treatment. Furthermore, we have also added tissues from younger animals to explore these interactions during post-natal development.