Recent studies highlight astrocytes as key drivers of motor neuron (MN) degeneration and disease propagation in mutant human superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis. However, in vivo analysis of specific astrocytic influence in amyotrophic lateral sclerosis has proven difficult because mSOD1 is ubiquitously expressed throughout the CNS of rodent models studied. Here, we transplanted SOD1^G93A glial-restricted precursor cells—glial progenitors capable of differentiating into astrocytes—into the cervical spinal cord of WT rats to reveal how mutant astrocytes influence WT MNs and other cells types (microglia and astrocytes) in an in vivo setting. Transplanted SOD1^G93A glial-restricted precursor cells survived and differentiated efficiently into astrocytes. Graft-derived SOD1^G93A astrocytes induced host MN ubiquitination and death, forelimb motor and respiratory dysfunction, reactive astrocytosis, and reduced GLT-1 transporter expression in WT animals. The SOD1^G93A astrocyte-induced MN death seemed in part mediated by host microglial activation. These findings show that mSOD1 astrocytes alone can induce WT MN death and associated pathological changes in vivo.