The mechanism(s) by which ionizing radiation (IR) induces cell death is of fundamental importance in understanding cell sensitivity and resistance. Here we evaluated the response to IR of two subclones of the autonomous human erythromyeloblastic cell line TF-1: TF-1-34 (which expresses CD34) and TF-1-33 (which lacks CD34). In clonogenic assays, TF-1-34 cells were found to be relatively less sensitive to IR compared to TF-1-33 cells based on the D0 determination (3.01 vs 1.56 Gy). Furthermore, after IR at 12 Gy, TF-1-33 cell viability decreased by approximately 50% within 24 h, whereas TF-1-34 cell growth was unaffected during this time. Gradual loss of TF-1-34 cell viability was observed only after 48 h. Morphological and molecular analysis revealed that TF-1-33 cells died of apoptosis, and TF-1-34 cells of delayed reproductive cell death. While IR produced comparable amounts of DNA double strand breaks (DSB) in both cell lines, TF-1-34 retained DSB much longer than TF-1-33 suggesting that radioresistance and the defective apoptotic response of TF-1-34 cells was not related to a higher DNA repair capacity. However, the lack of an apoptotic response in TF-1-34 was correlated to the absence of a sphingomyelin (SM)-ceramide (CER) signaling pathway. Indeed, IR triggered in TF-1-33 cells but not in TF-1-34, SM hydrolysis (25% at 12 Gy) and CER generation (>50%) through the activation of neutral but not acid sphingomyelinase. Synthetic cell permeate CER induced apoptosis in both TF-1-33 and TF-1-34 cells. This study indicates that alterations of the SM-CER signaling pathway can significantly influence the cell death process as well as the survival of acute myeloid leukemia cells after IR exposure.