Current advances allow for efficient generation of neuron, astrocyte and microglia cultures from human pluripotent stem cells (hPSCs). However, monocultures lack physiologically important glia-neuron interactions. We created a protocol to co-culture neurons with astrocytes and microglia to assess the impact of glia on neuronal morphology and recovery under injury conditions. We generated hPSC-derived forebrain neurons, astrocytes and microglia using the STEMdiff™ Forebrain Neuron, Astrocyte and Microglia culture systems, respectively. We maintained neuron-astrocyte co-cultures for one week using STEMdiff™ Forebrain Neuron Maturation Medium. We evaluated the effect of astrocyte presence on neuronal phenotypes by measuring neurite length and number of MAP2-positive cells. Next, we established a tri-culture model containing hPSC-derived neurons, astrocytes and microglia in BrainPhys™-based hPSC Neuronal Medium with STEMdiff™ Microglia Supplement 2. To assess response to injury, we scratched the cultures and measured the percentage of area covered by GFAP+ astrocytes, βIIITUB+ neurons and GFP-fluorescent microglia after 48 hours. After one week of neuron-astrocyte co-culture, we observed a non-significant increase in MAP2+ neurons and a significant 1.26 ± 0.09-fold increase in neurite length (mean ± SEM, normalized to control; n = 4 - 5; p = 0.04) for co-cultured neurons. Preliminary results suggest that microglia in the tri-culture may positively correlate to an increase of βIIITUB+ neurons in the injury area (n = 1, p < 0.05). These results demonstrate that populations of astrocytes, neurons and microglia can be cultured together to display developed neuronal phenotypes and functional properties compared to neuron monocultures.