Two related receptor tyrosine kinases (RTKs), fibroblast growth factor receptor 1 and 2 (FGFR1 and FGFR2), exert distinct effects during carcinogenesis. To examine FGFR1 and FGFR2 signaling in polarized epithelia, we have developed an in vitro three-dimensional HC11 mouse mammary epithelial cell culture model combined with a chemically inducible FGFR (iFGFR) dimerization system. Although activation of both RTKs led to reinitiation of cell proliferation and loss of cell polarity, only iFGFR1 activation induced cell survival and epithelial to mesenchymal transition. In contrast, iFGFR2 activation induced cell apoptosis even in the cells in direct contact with the extracellular matrix. Activation of iFGFR2, but not iFGFR1, led to rapid receptor down-regulation and transient activation of downstream signaling, which were partially rescued by Cbl small interfering RNA knockdown or the proteasome inhibitor lactacystin. Importantly, inhibition of proteasome activity in iFGFR2-activated structures led to epithelial to mesenchymal transition and invasive phenotypes resembling those observed after iFGFR1 activation. These studies demonstrate, for the first time, that the duration of downstream signaling determines the distinct phenotypes mediated by very homologous RTKs in three-dimensional cultures.