Although hyperactivation of Ras is a common feature of myeloid malignancy, its role in subverting hematopoiesis is unclear. We have examined the influence of Ras on normal human uncommitted myeloid subsets and show that expression of this oncogene strongly favors monocyte lineage selection in bipotential granulocyte/macrophage progenitors while inhibiting colony formation in other uncommitted subsets. Ras also promoted monocytic differentiation but not the proliferation of these cells. The mechanism through which Ras drives monocyte lineage selection was dependent on PKC activity and Ras was found to promote the expression, membrane translocation, and phosphorylation of conventional and novel PKC isoforms. We further show that Ras promoted the expression of the AGC kinase master regulator, PDK1, which maintains the stability and activity of PKC isoforms. Consistent with this, overexpression of PDK1 itself promoted monocyte colony formation and translocation of PKC. Overexpression of PDK1 was found to be a common feature of acute myeloid leukemia (45% of patients) and was closely associated with hyperphosphorylation of PKC. These data demonstrate that Ras is able to promote monocyte lineage selection via PKC and show for the first time the involvement of the kinase master regulator, PDK1, in both lineage specification and in human leukemia.