The absence of ectoderm impairs somite formation in cultured presomitic mesoderm (PSM) explants, suggesting that an ectoderm-derived signal is essential for somitogenesis. Here we show in chick that the standard enzymatic treatments used for explant isolation destroy the fibronectin matrix surrounding the anterior PSM, which fails to form somites when cultured for 6 hours. By contrast, explants isolated with collagenase retain their fibronectin matrix and form somites under identical culture conditions. The additional presence of ectoderm enhances somite formation, whereas endoderm has no effect. Furthermore, we show that pancreatin-isolated PSM explants cultured in fibronectin-supplemented medium, form significantly more somites than control explants. Interestingly, ectoderm is the major producer of fibronectin (Fn1) transcripts, whereas all but the anterior-most region of the PSM expresses the fibronectin assembly receptor, integrin alpha5 (Itga5). We thus propose that the ectoderm-derived fibronectin is assembled by mesodermal alpha5beta1 integrin on the surface of the PSM. Finally, we demonstrate that inhibition of fibronectin fibrillogenesis in explants with ectoderm abrogates somitogenesis. We conclude that a fibronectin matrix is essential for morphological somite formation and that a major, previously unrecognised role of ectoderm in somitogenesis is the synthesis of fibronectin.