Background: Fibronectin (Fn) forms a centripetal gradient during the fetal adrenal gland organogenesis, and modulates hormone responsiveness of adrenocortical cells in the primary culture. However, how Fn is involved in organ formation of the adrenal gland remains unclear. Results: In this study, we found that Fn accumulates around migrating ff1b-expressing interrenal cells, which were marked by the ff1b promoter-driven transgenic fluorescence, during the course of interrenal organ assembly. The interrenal cells displaying the migratory phenotype were absent in the fn1 mutant, while specification and kidney association of the interrenal tissue remained normal. The Fn deposition in the interrenal microenvironment was severely reduced in the vessel-deficient ets1b morphant, implying its origin of synthesis from the peri-interrenal vasculature. In the fn1 mutant, early-migrating chromaffin cells were capable of interacting with steroidogenic interrenal cells, yet continuous migration and midline convergence of chromaffin cells were disrupted. Migration defects of both interrenal and chromaffin lineages, in the absence of Fn, thus led to incomplete interrenal organ assembly in aberrant positions. Conclusions: Our results indicate that Fn is essential for patterning interrenal organ formation, by modulating the migratory behavior of both steroidogenic interrenal and chromaffin cells. Developmental Dynamics 242:432–443, 2013. © 2013 Wiley Periodicals, Inc.