Abstract Ependymoma (EPN) is fatal in over 50% of children and has not seen any therapeutic improvements in over 30 years. Based on data from single-cell RNA sequencing (scRNAseq) and large-scale screening of FDA-approved oncology compounds in posterior fossa group A (PFA) EPN, we hypothesized that combination of PDGFR inhibitors and retinoids therapy will selectively deplete aggressive mesenchymal and undifferentiated EPN subpopulations resulting in therapeutic benefit. In vitro screening of a panel of three receptor PDGFR inhibitors (PDGFRIs) and nine retinoid analogues in high-risk chromosome 1q+/6q- PFA cell lines MAF-811 and MAF-928 was performed to identify those drugs with (i) anti-proliferative effect (tritiated thymidine incorporation and live-cell imaging), (ii) depletion of high-risk associated neoplastic subpopulations (qRT-PCR using subpopulation specific markers and scRNAseq), and (iii) synergistic effects. PDGFRIs axitinib and pazopanib and retinoids tazarotene, adapalene and tretinoin showed superior potency versus other analogues. qRT-PCR showed that tazarotene and axitinib demonstrated beneficial subpopulation perturbation. Combination of the top two PDGFRIs (axitinib and pazopanib) and retinoids (tazarotene and tretinoin) demonstrated that axitinib combined with tretinoin provided the strongest synergistic anti-proliferative effect and depletion of high-risk-associated undifferentiated subpopulations. More definitive scRNAseq subpopulation analysis revealed a reduction of mesenchymal cells in MAF-811 in response to combined axitinib/tretinoin treatments. In both MAF-811 and MAF-928 there was a net increase in the proportion of differentiated subpopulations, which are associated with a more favorable clinical outcome, in response to combined axitinib/tretinoin, collectively supporting our original hypothesis. These results demonstrate that combined RTKi axitinib and retinoid tretinoin treatment has a synergistic anti-proliferative effect and reduction of high-risk subpopulation proportions. The effect of combined axitinib and tretinoin on survival and subpopulation perturbation is now being explored in vivo.