Neurons in nucleus laminaris (NL) receive binaural, tonotopically matched input from nucleus magnocelluaris (NM) onto bitufted dendrites that display a gradient of dendritic arbor size. These features improve computation of interaural time differences, which are used to determine the locations of sound sources. The dendritic gradient emerges following a period of significant reorganization at embryonic day 15 (E15), which coincides with the emergence of astrocytes that express glial fibrillary acidic protein (GFAP) in the auditory brainstem. The major changes include a loss of total dendritic length, a systematic loss of primary dendrites along the tonotopic axis, and lengthening of primary dendrites on caudolateral NL neurons. Here we have tested whether astrocyte-derived molecules contribute to these changes in dendritic morphology. We used an organotypic brainstem slice preparation to perform repeated imaging of individual dye-filled NL neurons to determine the effects of astrocyte-conditioned medium (ACM) on dendritic morphology. We found that treatment with ACM induced a decrease in the number of primary dendrites in a tonotopically graded manner similar to that observed during normal development. Our data introduce a new interaction between astrocytes and neurons in the auditory brainstem and suggest that these astrocytes influence multiple aspects of auditory brainstem maturation.