Fibronectins (FNs) are major cell-adhesive proteins in the extracellular matrix and are essential for embryonic development. FNs are encoded by a single gene, but heterogeneity is introduced by alternative pre-mRNA splicing. One of the alternatively spliced segments, extra domain B (EDB), is prominently expressed during embryonic development and in tumor tissues, although it is mostly eliminated from FN in normal adult tissues. To examine the function of the EDB segment in vivo, we generated mice lacking the EDB exon using the Cre-loxP system. Although EDB-containing FNs are highly expressed throughout early embryogenesis, EDB-deficient mice developed normally and were fertile. Despite the absence of any significant phenotypes observed in vivo, however, fibroblasts obtained from EDB-deficient mice grew slowly in vitro and deposited less FN in the pericellular matrix than fibroblasts from wild-type mice. These results indicate that expression of EDB-containing isoforms is dispensable during embryonic development, yet may play a modulating role in the growth of connective tissue cells via the FN matrix.