Central nervous system injury often leads to functional impairment due, in part, to the formation of an inhibitory glial scar following injury that contributes to poor regeneration. Astrocytes are the major cellular components of the glial scar, which has led to the belief that they are primarily inhibitory following injury. Recent work has challenged this by demonstrating that some astrocytes are required for spinal cord regeneration and astrocytic roles in recovery depend on their phenotype. In this work, two mixed populations containing primarily either fibrous or protoplasmic astrocytes were derived from mouse embryonic stem cells (mESCs). Motoneuron and V2a interneuron growth on live cultures, freeze-lysed cultures, or decellularized extracellular matrix (ECM) from astrocytes were assessed. Both neuronal populations were found to extend significantly longer neurites on protoplasmic-derived substrates than fibrous-derived substrates. Interestingly, neurons extended longer neurites on protoplasmic-derived ECM than fibrous-derived ECM. ECM proteins were compared with in vivo astrocyte expression profiles, and it was found that the ESC-derived ECMs were enriched for astrocyte-specific proteins. Further characterization revealed that protoplasmic ECM had significantly higher levels of axon growth promoting proteins, while fibrous ECM had significantly higher levels of proteins that inhibit axon growth. Supporting this observation, knockdown of spondin-1 improved neurite growth on fibrous ECM, while laminin α5 and γ1 knockdown decreased neurite growth on protoplasmic ECM. These methods allow for scalable production of specific astrocyte subtype-containing populations with different neuronal growth support capacities, and can be used for further studies of the functional importance of astrocyte heterogeneity. © Copyright 2017, Mary Ann Liebert, Inc. 2017.