<p>Leukemic stem cells (LSCs) of acute myeloid leukemia (AML) can be enriched in the CD34⁺CD38^- fraction and reconstitute human AML in vivo. However, in acute promyelocytic leukemia (APL), which constitutes 10% of all AML cases and is driven by promyelocytic leukemia-retinoic acid receptor alpha (<i>PML::RARα</i>) fusion genes, the presence of LSCs has long been unidentified because of the difficulty in efficient reconstitution of human APL in vivo. Herein, we show that LSCs of the short-type isoform APL, a subtype of APL defined by different breakpoints of the <i>PML</i> gene, concentrate in the CD34⁺CD38⁻ fraction and express T cell immunoglobulin mucin-3 (TIM-3). Short-type APL cells exhibited distinct gene expression signatures, including LSC-related genes, compared to the other types of APL. Moreover, CD34⁺CD38⁻TIM-3⁺ short-type APL cells efficiently reconstituted human APL in xenograft models with high penetration, whereas CD34⁻ differentiated APL cells did not. Furthermore, CD34⁺CD38⁻TIM-3⁺ short-type APL cells reconstituted leukemia cells after serial transplantation. Thus, short-type APL was hierarchically organized by self-renewing APL-LSCs. The identification of LSCs in a subset of APL and establishment of an efficient patient-derived xenograft model may contribute to further understanding the APL leukemogenesis and devise individual treatments for the eradication of APL LSCs.</p>