Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture–initiating cells (LTC-ICs) directly isolated from patients are actively cycling, quiescent progenitors are present in most samples. In the current study,³H-thymidine (³H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G₀, G₁, and S/G₂+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the ³H-Tdr suicide results, with NOD/SL-ICs found almost exclusively among G₀ cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly, after 72 hours in serum-free culture with or without Steel factor (SF), Flt-3 ligand (FL), and interleukin-3 (IL-3), most G₀ AML cells entered active cell cycle (percentage of AML cells remaining in G₀ at 72 hours, 1.2% to 37%, and 0% to 7.6% in cultures without and with growth factors [GFs], respectively) while G₀ cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF, FL, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, 3 of 4 samples contained an internal tandem duplication of theFLT3 gene. In summary, quiescent leukemic cells, including NOD/SL-ICs, are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs.