Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34⁺ fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34⁺ fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34⁻ fraction, whereas the CD34⁺ fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34⁺ and CD34⁻ fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.