The Hedgehog (Hh) pathway regulates cell fate during metazoan development and adult tissue homeostasis by modulating tissue patterning, cell proliferation, and differentiation. In vertebrates, Hh signaling primarily relies on primary cilia. The pathway is activated when extracellular Sonic, Desert, or Indian Hedgehog ligands (Shh, Dhh, Ihh) bind to the transmembrane receptor Patched (Ptch) on Hedgehog-responsive cells. Ligand binding to Ptch allows the transmembrane protein Smoothened (Smo) to be transported to the primary cilium, where it activates the Gli transcription factors Gli2 and Gli3, which translocate to the nucleus and initiate the transcription of Hh target genes, including Gli1.