The activation of B-cell-specific genes, such as CD19 and PAX5, is a hallmark of t(8;21) acute myeloid leukemia (AML) which expresses the translocation product RUNX1/ETO. PAX5 is an important regulator of B-lymphoid development and blocks myeloid differentiation when ectopically expressed. To understand the molecular mechanism of PAX5 deregulation, we examined its chromatin structure and regulation in t(8;21) AML cells, non-t(8;21) myeloid precursor control cells, and pre-B cells. In non-t(8;21) myeloid precursors, PAX5 is poised for transcription, but is repressed by polycomb complexes. In t(8;21) AML, PAX5 is not directly activated by RUNX1/ETO, but expression requires constitutive mitogen-activated protein (MAP) kinase signaling. Using a model of t(8;21) carrying an activating KIT mutation, we demonstrate that deregulated MAP kinase signaling in t(8;21) AML abrogates the association of polycomb complexes to PAX5 and leads to aberrant gene activation. Our findings therefore suggest a novel role of activating tyrosine kinase mutations in lineage-inappropriate gene expression in AML.