MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88-/-, TLR2-/- and TLR4-/- mice. In MyD88-/- mice, the organogenesis of PPs was not disturbed. However, PPs in 2-week-old MyD88-/- mice were significantly smaller than those in MyD88+/- mice. Also, in 2-week-old TLR4-/-, but not TLR2-/- mice, PPs did not develop rapidly. The development of PPs in MyD88-/- and TLR4-/- mice was completely recovered in 10 weeks. PP cells from MyD88-/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10-week-old MyD88-/- mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.