Single-cell RNA-seq reveals a developmental hierarchy in human oligodendroglioma. Researchers used single-cell RNA sequencing to analyze 4,347 cells from six IDH1 or IDH2 mutant human oligodendrogliomas, identifying two specialized glial programs in most cancer cells and a rare undifferentiated subpopulation associated with a neural stem cell expression program. Cells with proliferation signatures were enriched in this subpopulation, suggesting cancer stem cells (CSCs) drive tumor growth. Copy number variation (CNV) analysis showed similar cellular hierarchies in distinct CNV subclones, indicating developmental programs dictate oligodendroglioma architecture. Subclonal point mutation analysis supported a similar model, though a full phylogenetic tree is needed to confirm genetic evolution's effect on hierarchies. Single-cell analyses provided insights into oligodendroglioma cellular architecture and supported the CSC model, with implications for disease management. Intra-tumoral heterogeneity contributes to therapy failure and cancer progression. While genetic evolution drives tumor heterogeneity, non-genetic programs like those in tissue stem cells and their differentiation also contribute. In human gliomas, candidate CSCs have been functionally isolated in high-grade lesions. However, functional approaches like in vivo xenotransplantation or in vitro sphere formation have generated controversy. It remains unknown if gliomas contain CSCs early in development, as in grade II lesions. A framework for analyzing cellular programs at single-cell resolution across genetic clones is critical. Oligodendroglioma, an incurable glioma with IDH1/IDH2 mutations and 1p/19q co-deletion, was studied using single-cell RNA-seq from six untreated grade II oligodendrogliomas. Analysis of 4,347 cells revealed a large majority with 1p/19q co-deletion and some tumor-specific CNVs. Two tumors had two subclones, and 303 cells lacked detectable CNVs, clustering into microglia and mature oligodendrocyte marker subsets. Analysis of three tumors with the largest cell numbers identified two prominent groups of cells, corresponding to low and high PC1 scores, expressing distinct lineage markers of astrocytes and oligodendrocytes. These results were consistent across all six tumors. Cells with high PC1 scores were strongly associated with high expression of 137 genes, including oligodendrocytic markers, and low expression of 128 genes, including astrocytic markers. Cells with low PC1 scores had the opposite patterns. This suggests oligodendrogliomas are primarily composed of two subpopulations of glial cells, mirroring histopathology. Cells with high PC2 and PC3 scores had intermediate PC1 scores, suggesting a lack of differentiation. A total of 63 genes were associated with high PC2 and PC3Single-cell RNA-seq reveals a developmental hierarchy in human oligodendroglioma. Researchers used single-cell RNA sequencing to analyze 4,347 cells from six IDH1 or IDH2 mutant human oligodendrogliomas, identifying two specialized glial programs in most cancer cells and a rare undifferentiated subpopulation associated with a neural stem cell expression program. Cells with proliferation signatures were enriched in this subpopulation, suggesting cancer stem cells (CSCs) drive tumor growth. Copy number variation (CNV) analysis showed similar cellular hierarchies in distinct CNV subclones, indicating developmental programs dictate oligodendroglioma architecture. Subclonal point mutation analysis supported a similar model, though a full phylogenetic tree is needed to confirm genetic evolution's effect on hierarchies. Single-cell analyses provided insights into oligodendroglioma cellular architecture and supported the CSC model, with implications for disease management. Intra-tumoral heterogeneity contributes to therapy failure and cancer progression. While genetic evolution drives tumor heterogeneity, non-genetic programs like those in tissue stem cells and their differentiation also contribute. In human gliomas, candidate CSCs have been functionally isolated in high-grade lesions. However, functional approaches like in vivo xenotransplantation or in vitro sphere formation have generated controversy. It remains unknown if gliomas contain CSCs early in development, as in grade II lesions. A framework for analyzing cellular programs at single-cell resolution across genetic clones is critical. Oligodendroglioma, an incurable glioma with IDH1/IDH2 mutations and 1p/19q co-deletion, was studied using single-cell RNA-seq from six untreated grade II oligodendrogliomas. Analysis of 4,347 cells revealed a large majority with 1p/19q co-deletion and some tumor-specific CNVs. Two tumors had two subclones, and 303 cells lacked detectable CNVs, clustering into microglia and mature oligodendrocyte marker subsets. Analysis of three tumors with the largest cell numbers identified two prominent groups of cells, corresponding to low and high PC1 scores, expressing distinct lineage markers of astrocytes and oligodendrocytes. These results were consistent across all six tumors. Cells with high PC1 scores were strongly associated with high expression of 137 genes, including oligodendrocytic markers, and low expression of 128 genes, including astrocytic markers. Cells with low PC1 scores had the opposite patterns. This suggests oligodendrogliomas are primarily composed of two subpopulations of glial cells, mirroring histopathology. Cells with high PC2 and PC3 scores had intermediate PC1 scores, suggesting a lack of differentiation. A total of 63 genes were associated with high PC2 and PC3