Three independent mutations were made by homologous recombination in two different regions of the fibronectin (FN) gene; all three appeared to be functional null mutations. The embryonic lethal phenotypes of these mutations were indistinguishable; all three FN mutant strains show mesodermal defects and fail to develop notochord or somites. Nevertheless analysis with lineage markers (Brachyury, sonic hedgehog, Notch-1, and mox-1) showed that both the notochord and the somite lineages were induced at the correct times and places. Furthermore, notochord precursor cells showed extensive cell migration in the absence of FN. However, neither notochord nor somites condensed properly in the absence of FN. These results show that specification of notochordal and somitic mesodermal lineages and significant cell migration are independent of fibronectin but that correct morphogenesis of these structures is FN-dependent.