Although growth factor signaling is required for embryonic development of organs, individual signaling mechanisms regulating these organotypic processes are just beginning to be defined. We compared signaling activated in fetal mouse submandibular glands (SMGs) by three growth factors, epidermal growth factor (EGF), fibroblast growth factor (FGF) 7, or FGF10, and correlated it with specific events of branching morphogenesis. Immunoblotting showed that EGF strongly stimulated phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and weakly stimulated phosphorylation of phospholipase Cgamma1 (PLCgamma1) and phosphatidylinositol-3 kinase (PI3K) in cultured E14 SMG. However, FGF7 and FGF10 stimulated phosphorylation of both PLCgamma1 and PI3K, but elicited only minimal phosphorylation of ERK-1/2. Morphological study of mesenchyme-free SMG epithelium cultured in Matrigel revealed that EGF induced cleft formation of endpieces, that FGF7 stimulated both cleft formation and stalk elongation, but that FGF10 induced only stalk elongation. In mesenchyme-free SMG epithelium cultured with EGF, FGF7 and FGF10, U0126 (MEK inhibitor) completely blocked cleft formation, whereas U73122 (PLCgamma1 inhibitor) suppressed stalk elongation. These finding suggest that EGF stimulates cleft formation and drives branch formation via ERK-1/2, and that FGF7 stimulates both cleft formation and stalk elongation via PLCgamma1 and partly via ERK-1/2, but that FGF10 stimulates stalk elongation mainly via PLCgamma1.