SummaryHepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and causing them to migrate as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. We have applied a chemical biology approach to identify components of HGF signaling that are required for scattering in an in vitro model system. This approach yields a number of small molecules that block HGF-induced scattering, including a calcium channel blocker. We show that HGF stimulation results in sudden and transient increases in ion channel influxes at the plasma membrane. Although multiple channels occur in the membranes of our model system, we find that TrpC6 is specifically required for HGF-induced scattering. We further demonstrate that HGF-induced ion influxes through TrpC6 channels coincide with a transient increase in nuclear factor of activated T-cells (NFAT)-dependent gene transcription and that NFAT-dependent gene transcription is required for HGF-induced cell scattering.