M cells are phagocytic intestinal epithelial cells in the follicle-associated epithelium (FAE) of Peyer's patches (PPs) that transport particulate antigens from the gut lumen into the subepithelial dome. Differentiation of M cells from epithelial stem cells in intestinal crypts requires the cytokine RANKL and the transcription factor Spi-B. We used three-dimensional enteroid cultures established with small intestinal crypts from mice as a model system to investigate signaling pathways involved in M cell differentiation and the influence of other cytokines on RANKL-induced M cell differentiation. RANKL addition to enteroids induced expression of multiple M-cell-associated genes by 1 day, including Spib, Ccl9, Tnfaip2, Anxa5 and Marcksl1. The mature M-cell marker Gp2 was strongly induced by 3 days and expressed by 11% of cells in enteroids. The noncanonical NF-B pathway was required for RANKL-induced M cell differentiation in enteroids, as addition of RANKL to enteroids from mice with a null mutation in the Map3k14 gene encoding NF-B-inducing kinase failed to induce M-cell-associated genes. While the cytokine TNF-α alone had little if any effect on expression of M-cell-associated genes, adding TNF-α to RANKL consistently resulted in 3- to 6-fold higher levels of multiple M-cell-associated genes compared to RANKL alone. One contributing mechanism is the rapid induction by TNF-α of Relb and Nfkb2, genes encoding the two subunits of the noncanonical NF-B heterodimer. We conclude that endogenous activators of canonical NF-B signaling present in the GALT microenvironment including TNF-α can play a supportive role in the RANKL-dependent differentiation of M cells in the FAE.