The rapid rise of chemical biology aimed at studying signaling networks for basic cellular activities using specific, active small molecules as probes has greatly accelerated research on pathological mechanisms and target therapy of diseases. This research is especially important for malignant tumors such as leukemia, a heterogeneous group of hematopoietic malignancies that occurs worldwide. With the use of a chemical approach combined with genetic manipulation, great progress has been achieved over the past few decades on the biological, molecular and cytogenetic aspects of leukemia, and in its diagnosis and therapy. In particular, discoveries of the clinical effectiveness of all-trans retinoic acid and arsenic trioxide in the treatment of acute promyelocytic leukemia and the kinase inhibitors Imatinib and Dasatinib in the treatment of chronic myelogenous leukemia not only make target therapy of leukemia a reality, but also push mechanisms of leukemogenesis and leukemic cell activities forward. This review will outline advances in chemical biology that help our understanding of the molecular mechanisms of cell differentiation and apoptosis induction and target therapy of leukemia.