To study mechanisms that regulate the construction of inhibitory circuits, we examined the role of brain-derived neurotrophic factor (BDNF) in the assembly of GABAergic inhibitory synapses in the mouse cerebellar cortex. We show that within the cerebellum, BDNF-expressing cells are restricted to the internal granular layer (IGL), but that the BDNF protein is present within mossy fibers which originate from cells located outside of the cerebellum. In contrast to deletion of TrkB, the cognate receptor for BDNF, deletion of Bdnf from cerebellar cell bodies alone did not perturb the localization of pre- or postsynaptic constituents at the GABAergic synapses formed by Golgi cell axons on granule cell dendrites within the IGL. Instead, we found that BDNF derived from excitatory mossy fiber endings controls their differentiation. Our findings thus indicate that cerebellar BDNF is derived primarily from excitatory neurons--precerebellar nuclei/spinal cord neurons that give rise to mossy fibers--and promotes GABAergic synapse formation as a result of release from axons. Thus, within the cerebellum the preferential localization of BDNF to axons enhances the specificity through which BDNF promotes GABAergic synaptic differentiation.