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Single prostate stem cells can generate stem and progenitor cells to form prostaspheres in 3D culture. Using a prostasphere-based label retention assay, we recently identified keratin 13 (<i>KRT13</i>)-enriched prostate stem cells at single-cell resolution, distinguishing them from daughter progenitors. Herein, we characterized the epithelial cell lineage hierarchy in prostaspheres using single-cell RNA-seq analysis. Keratin profiling revealed three clusters of label-retaining prostate stem cells; <i>cluster I</i> represents quiescent stem cells (<i>PSCA</i>, <i>CD36</i>, <i>SPINK1</i>, and <i>KRT13/23/80/78/4</i> enriched), while <i>clusters II</i> and <i>III</i> represent active stem and bipotent progenitor cells (<i>KRT16/17/6</i> enriched). Gene set enrichment analysis revealed enrichment of stem and cancer-related pathways in <i>cluster I</i>. In non-label-retaining daughter progenitor cells, three clusters were identified; <i>cluster IV</i> represents basal progenitors (<i>KRT5/14/6/16</i> enriched), while <i>clusters V</i> and <i>VI</i> represent early and late-stage luminal progenitors, respectively (<i>KRT8/18/10</i> enriched). Furthermore, MetaCore analysis showed enrichment of the “cytoskeleton remodeling–keratin filaments” pathway in cancer stem-like cells from human prostate cancer specimens. Along with common keratins (<i>KRT13/23/80/78/4</i>) in normal stem cells, unique keratins (<i>KRT10/19/6C/16</i>) were enriched in cancer stem-like cells. Clarification of these keratin profiles in human prostate stem cell lineage hierarchy and cancer stem-like cells can facilitate the identification and therapeutic targeting of prostate cancer stem-like cells.