Spinal muscular atrophy (SMA) is a motoneuron disease caused by loss or mutation in Survival of Motor Neuron 1 (SMN1) gene. Recent studies have shown that selective restoration of SMN protein in astrocytes partially alleviates pathology in an SMA mouse model, suggesting important roles for astrocytes in SMA. Addressing these underlying mechanisms may provide new therapeutic avenues to fight SMA. Using primary cultures of pure motoneurons or astrocytes fromSMNΔ7(SMA) and wild-type (WT) mice, as well as their mixed and matched cocultures, we characterized the contributions of motoneurons, astrocytes, and their interactions to synapse loss in SMA. In pure motoneuron cultures, SMA motoneurons exhibited normal survival but intrinsic defects in synapse formation and synaptic transmission. In pure astrocyte cultures, SMA astrocytes exhibited defects in calcium homeostasis. In motoneuron–astrocyte contact cocultures, synapse formation and synaptic transmission were significantly reduced when either motoneurons, astrocytes or both were from SMA mice compared with those in WT motoneurons cocultured with WT astrocytes. The reduced synaptic activity is unlikely due to changes in motoneuron excitability. This disruption in synapse formation and synaptic transmission by SMN deficiency was not detected in motoneuron–astrocyte noncontact cocultures. Additionally, we observed a downregulation of Ephrin B2 in SMA astrocytes. These findings suggest that there are both cell autonomous and non–cell-autonomous defects in SMA motoneurons and astrocytes. Defects in contact interactions between SMA motoneurons and astrocytes impair synaptogenesis seen in SMA pathology, possibly due to the disruption of the Ephrin B2 pathway.SIGNIFICANCE STATEMENTAstrocytes have been implicated in various neurological diseases, including spinal muscular atrophy (SMA), a childhood motoneuron disease. However, how bidirectional motoneuron–astrocyte communications may contribute to SMA disease mechanisms is unclear. Usingin vitroculture systems, we demonstrate that SMN deficiency affects both astrocytes and motoneurons, as well as their contact interactions, leading to defects in synapse formation and synaptic transmission onto motoneurons, probably through downregulation of Ephrin B2 expression in astrocytes. Our work suggests that defects in motoneuron–astrocyte interactions exacerbate SMA synaptic pathology and that therapeutic restoration of SMN should target multiple cell types, including astrocytes.