The protein kinase C (PKC) family of serine/threonine kinases are pleiotropic signaling regulators and are implicated in hematopoietic signaling and development. Only one isoform however, PKCϵ, has oncogenic properties in solid cancers where it is associated with poor outcomes. Here we show that PKCϵ protein is significantly overexpressed in acute myeloid leukemia (AML; 37% of patients). In addition, PKCϵ expression in AML was associated with a significant reduction in complete remission induction and disease-free survival. Examination of the functional consequences of PKCϵ overexpression in normal human hematopoiesis, showed that PKCϵ promotes myeloid differentiation, particularly of the monocytic lineage, and decreased colony formation, suggesting that PKCϵ does not act as an oncogene in hematopoietic cells. Rather, in AML cell lines, PKCϵ overexpression selectively conferred resistance to the chemotherapeutic agent, daunorubicin, by reducing intracellular concentrations of this agent. Mechanistic analysis showed that PKCϵ promoted the expression of the efflux pump, P-GP (ABCB1), and that drug efflux mediated by this transporter fully accounted for the daunorubicin resistance associated with PKCϵ overexpression. Analysis of AML patient samples also showed a link between PKCϵ and P-GP protein expression suggesting that PKCϵ expression drives treatment resistance in AML by upregulating P-GP expression.