Interferon alfa (IFN-α) is approved for the therapy of patients with polycythemia vera (PV), a subtype of myeloproliferative neoplasm (MPN). Some patients achieve molecular responses (MRs), but clonal factors sensitizing for MRs remain elusive. We integrated colony formation assays with single-cell RNA sequencing (scRNA-seq) and genotyping in PV-derived cells and healthy controls (HCs) to dissect how IFN-α targets diseased clones during erythroid differentiation. IFN-α significantly decreased colony growth in MPNs and HCs with variable transcriptional responses observed in individual colonies. scRNA-seq of colonies demonstrated more mature erythroid colonies in PV than HCs. JAK2V617F-mutant cells exhibited upregulated STAT5A, heme, and G2M checkpoint pathways compared with JAK2WT cells from the same patients. Subgroup analysis revealed that IFN-α significantly decreased immature erythrocytic cells in PV (basophilic erythroblasts P < .05; polychromatic erythroblasts P < .05) but not in HCs. CD71-/CD235a+ cells from HCs (P < .05) but not PV were inhibited by IFN-α, and the number of reticulocytes was less affected in PV. Robust IFN-α responses persisted throughout differentiation, leading to significant apoptosis in PV. Apoptotic cells displayed downregulation of ribosomal genes. This link between apoptosis and ribosomal genes was corroborated through the analysis of mitochondrial variants, demonstrating IFN-α-induced eradication of specific clones, characterized by elevated expression of ribosomal genes. Our findings indicate that PV-derived clones either undergo apoptosis or pass through differentiation, overall reducing the cycling mutant cells over long-term treatment. Furthermore, the significance of ribosomal genes and clonal prerequisites in IFN-α's therapeutic mechanism is underscored, shedding light on the intricate dynamics of IFN-α treatment in PV.