Though the zebrafish interrenal tissue has been used as a model for steroidogenesis and genesis of the adrenal gland, its specification and morphogenesis is still largely unclear. In this study, we explored how the WT1-expressing cells are segregated from the SF-1-expressing steroidogenic cells in the zebrafish model. The interrenal tissue precursors expressing ff1b, the equivalent of mammalian SF-1, are derived from wt1-expressing pronephric primordia in the zebrafish embryo. Through histochemistry and in situ hybridization, we demonstrated that the size of functionally-differentiated interrenal tissue was significantly increased upon a global inhibition of the Notch signaling pathway, which was accompanied by a disrupted segregation between the wt1- and ff1b-expressing cells. While the Notch pathway was conditionally activated during the interrenal specification, differentiation but not ff1b-expression of interrenal tissue was drastically compromised. In embryos deficient for Notch ligands jagged 1b and 2b, transgenic reporter activity of wt1b promoter was detected within the steroidogenic interrenal tissue. In summary, our results indicate that Jagged-Notch signaling is required for (1) the segregation between wt1-expressing cells and differentiated steroidogenic tissue; and (2) modulating the extent of functional differentiation in the steroidogenic interrenal tissue.