"There is little evidence to demonstrate the importance of the Sonic hedgehog homolog (Shh) pathway to differentiation therapy in the treatment of hematological neoplasms. Here we characterize the changes in acute myelogenous leukemia (HL-60) cells after blocking the Shh pathway by an antagonist of Smoothened, cyclopamine. Cyclopamine induces apoptosis of HL-60 cells in a dose- and time-dependent manner with increased G0/G1 cycle fraction. Treatment with cyclopamine increases the expression of monocytic cell markers CD11b and CD14, but the expression of CD13, CD33 and CD38 is unchanged. The monocytic differentiation of HL-60 cells induced by cyclopamine is also evidenced by an increase in Egr-1 expression. Importantly, cyclopamine down-regulates the phosphorylation of Akt and ERK, but activates AMP-activated protein kinase (AMPK) signaling. Further investigations should determine the clinical application of modulating the Shh pathway in the treatment of hematological malignancies.