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M cells are specialized epithelial cells, which assist immune surveillance by transcytosis of particles and antigens to underlying lymphoid tissues. So far, three M cell phenotypes have been identified in airways and intestines. However, the mechanism of M cell differentiation is poorly understood, as well as the relationships between different M cell subtypes. To better understand these questions, we treated human (Caco-2BBe) and rodent (IEC-6) intestinal epithelial cell lines with lymphotoxin beta receptor (LTbetaR) and TNF receptor (TNFR) agonists. Treated cells were then studied for regulation of genes previously shown to associate with M cell and Follicle-Associated Epithelium (FAE). Moreover, we used a reporter transgenic mouse strain to follow the development of M cell subsets in vivo, and examined the de novo induction of M cells by cholera toxin (CT).We found that LTbetaR and TNFR agonists can induce transcription of FAE specific genes (Ccl20 and Lamb3) as well as rodent M cell specific genes such as Sgne-1/Scg5, Cldn4, and Gp2 in Caco2-BBe cells and IEC-6 cells. These cytokines have distinct but complementary effects; TNFR agonists induced FAE specific genes, while the LTbetaR agonist induces M cell specific genes. The combination of these cytokines showed additive induction of the FAE-associated Ccl20, Lamb3 and a surprisingly the induction of CD137/Tnfrsf9. Functionally, cytokine treatment led to both the reorganization of microvilli as well as Claudin-4 redistribution. Moreover, with the help of the reporter transgenic mouse model, we have found that M cells overlying intestinal Peyer's patches (PP) and Nasal Associated Lymphoid Tissue (NALT) have distinct developmental origins, yet show convergent phenotypes, such as expression of a PGRP-S reporter in vivo. By contrast, though PP and Villous M cells are derived from intestinal crypt stem cells, their phenotypes are clearly distinct. Thus, PP M cells form an intimate M cell-dendritic cell functional unit while Villous M cells do not engage with underlying DC. Though B lymphocytes are thought to play a critical role in M cell function by forming a basolateral pocket and possibly signaling through CD137, initial commitment to M cell lineages is B lymphocyte and CD137 independent. In addition, CT can causes rapid induction of new M cells in the airway and intestines without cell division, suggesting transdifferentiation from mature epithelial cells. CT-induced M cells show the local M cell phenotype without evidence for mixed phenotypes. Finally, M cell phenotypes display distinct capabilities; thus, intestinal PP M cells were more efficient than Villous M cells in the uptake of Salmonella. Our studies show that LTbetaR and TNFR agonists induce many features associated with M cell differentiation, and suggest that these agonists may be involved in secondary lymphoid tissue development in vivo. Additionally, we confirmed that the differentiation of M cells in vivo toward strictly defined phenotypic subsets, and is consistent with functional specialization.