We recently described oncogenic and anti-apoptoticC-RAFgermline mutations in patients with therapy-related acute myeloid leukemia (t-AML). Activation of the RAF effector ERK was restricted to transformed cells, suggesting the requirement for cooperating events in leukemogenesis. Western blot analysis of blast cells from patients withC-RAFgermline mutations revealed loss of the tumor and metastasis suppressor RAF kinase inhibitor protein (RKIP). Immunohistochemistry of the patients' primary tumors revealed normal RKIP expression levels, indicating that the loss of RKIP is a somatic, t-AML-specific event. In focus formation assays, the oncogenic potential of human mutant C-RAF was strongly influenced by expression levels of RKIP. Although the number of colonies formed by C-RAFS427G was significantly increased by RKIP silencing, the opposite was observed after RKIP overexpression. These results show that the loss of RKIP is a functional somatic event in carriers ofC-RAFgermline mutations, which contributes to the development of t-AML.