Acute myeloid leukemia (AML) is an aggressive and heterogeneous disease characterized by an abnormal proliferation and impaired differentiation of the myeloid precursor cells. The outcome for most AML patients remains poor with high relapse rates and chemotherapy remains the first line treatment for AML. The Wilms tumor wt1 and the anti-apoptotic BCL2 genes are upregulated in AML and are known to be involved in apoptosis inhibition. In the present study we evaluated the molecular mechanisms underlie the anti-proliferative and pro-apoptotic activities exerted by thymoquinone (TQ), the major biologically active compound of the black seed oil on acute myeloid leukemia (AML) cell line-HL60. Cell proliferation was determined by WST-1 assay and apoptosis rate was assessed by flow cytometry using annexin-V/7AAD staining. The expression of target genes was analyzed by real-time RT–PCR analysis. TQ significantly reduced HL60 cell viability and induced apoptosis in a dose and time-dependent manner. In order to decipher the molecular mechanisms underlie the anti-cancer activities induced by TQ in AML cells, we investigated its effect on the expression of WT1 and BCL2 genes. TQ significantly decreased the expression of WT1 and BCL2 genes in a dose and time-dependent manner. In summary, these findings suggest that TQ induces cell proliferation inhibition and apoptosis in acute myeloid leukemia cells most likely through targeting the apoptosis-related WT1 and BCL2 genes and also suggest that TQ could be a promising strategy for AML therapy.