The Hedgehog (HH) pathway regulates cell proliferation and survival and contributes to tumorigenesis. However, not much is known about the role of the HH-signaling system in the bone marrow microenvironment during the development of Acute Myeloid Leukemia (AML). The aim of this study was to investigate the anti-leukemic effects of the HH-signaling pathway and molecular mechanisms of apoptosis induction in AML cells, by blocking the hedgehog signaling pathway during co-culture of AML cells with Bone-Marrow Stromal Cells (BMSCs). It was found that the HS-5 line of BMSCs protected AML cells from spontaneous apoptosis via up-regulation of transcription factors GLI1, BCL-2 and BCL-XL. These results indicate that the up-regulation of the transcription factors involved activation of the hedgehog pathway. Thus, targeting a microenvironment-related signaling pathway may be a novel approach to AML therapy.