The glial tube is a longitudinal structure predominantly composed of densely bundled, aligned astrocytes that projects from the subventricular zone (SVZ) to the olfactory bulb. Neural precursor cells (NPCs) generated in the SVZ migrate through this glial tube—referred to as the rostral migratory stream (RMS)—to replace olfactory bulb interneurons in the mammalian brain. RMS astrocytes have distinct morphological and functional characteristics. These characteristics facilitate the unique purpose of the RMS as an endogenous living scaffold directing NPC migration and maturation. However, the transcriptomic factors underlying these unique structure–function attributes versus standard stellate astrocytes have not been examined. We previously developed biofabrication techniques to create the first tissue-engineered rostral migratory stream (TE-RMS) that replicates key features of the glial tube in vivo. We have shown that TE-RMS astrocytes exhibit elongated nuclei, longitudinally aligned intermediate filaments, and enrichment of key functional proteins—cytoarchitectural and surface features characteristic of native RMS astrocytes. In the current study, we performed RNA-seq on TE-RMS astrocytes in comparison to planar astrocyte cultures to identify gene expression patterns that may underlie their profound morphological and functional differences. Remarkably, we found 4,008 differentially expressed genes in TE-RMS astrocytes, with 2076 downregulated (e.g., LOC690251 and ccn5) and 1932 upregulated (e.g., lrrc45 and cntn1) compared to planar astrocytes. Moreover, there were 256 downregulated and 91 upregulated genes with >3-fold change. We also conducted analyses of gene sets related to cytoskeleton and nuclear structure, revealing the greatest enrichment of actin-related components. Overall, the TE-RMS offers a platform to study the interplay between transcriptomic and cytoarchitectural dynamics in a unique astrocyte population.