AbstractGrafting is a plant propagation technique widely used in agriculture. A recent discovery of the capability of interfamily grafting inNicotianahas expanded the potential combinations of grafting. In this study, we showed that xylem connection is essential for the achievement of interfamily grafting and investigated the molecular basis of xylem formation at the graft junction. Transcriptome and gene network analyses revealed gene modules for tracheary element (TE) formation during grafting that include genes associated with xylem cell differentiation and immune response. The reliability of the drawn network was validated by examining the role of theNicotiana benthamiana XYLEM CYSTEINE PROTEASE(NbXCP) genes in TE formation during interfamily grafting. Promoter activities ofNbXCP1andNbXCP2genes were found in differentiating TE cells in the stem and callus tissues at the graft junction. Analysis of aNbxcp1;Nbxcp2loss-of-function mutant indicated thatNbXCPscontrol the timing ofde novoTE formation at the graft junction. Moreover, grafts of theNbXCP1overexpressor increased the scion growth rate as well as the fruit size. Thus, we identified gene modules for TE formation at the graft boundary and demonstrated potential ways to enhanceNicotianainterfamily grafting.