RUNX1 (also known as AML1) is a DNA-binding transcription factor that functions as a tumor suppressor and developmental determinant in hematopoietic cells. Target promoters have been identified primarily through the use of differential expression strategies and candidate gene approaches but not biochemical screens. Using a chromatin immunoprecipitation screen, we identified protein kinase Cbeta as a direct RUNX1 target gene and demonstrate that endogenous RUNX1 binds the chromatinized protein kinase Cbeta promoter of U937 cells. A phylogenetically conserved RUNX1-binding site within the PKCbeta promoter binds RUNX1 in electrophoretic mobility shift analyses and confers RUNX1 responsiveness on a heterologous promoter. Changes in RUNX1 activity affect endogenous protein kinase Cbeta expression, and a dominant-negative form of RUNX1 protects U937 cells from apoptotic stimuli previously shown to be dependent on protein kinase Cbeta. This protection can be reversed by the ectopic expression of protein kinase Cbeta. Together these findings demonstrate that protein kinase Cbeta is a direct, downstream target of RUNX1 and links RUNX1 to a myeloid apoptotic pathway.