Target-derived cues promote local differentiation of axons into nerve terminals at sites of synaptic contact. Using clustering of synaptic vesicles in cultured neurons as an assay, we purified putative target-derived presynaptic organizing molecules from mouse brain and identified FGF22 as a major active species. FGF7 and FGF10, the closest relatives of FGF22, share this activity; other FGFs have distinct effects. FGF22 is expressed by cerebellar granule cells during the period when they receive synapses. Its receptor, FGFR2, is expressed by pontine and vestibular neurons when their axons (mossy fibers) are making synapses on granule cells. Neutralization of FGF7, -10, and -22 inhibits presynaptic differentiation of mossy fibers at sites of contact with granule cells in vivo. Inactivation of FGFR2 has similar effects. These results indicate that FGF22 and its relatives are presynaptic organizing molecules in the mammalian brain and suggest new functions for this family of signaling molecules.