Pim2 is a serine/threonine kinase expressed at high levels in several malignancies including acute leukemia. Pim2 protein is induced by oncogenic Fms-like tyrosine kinase-3 (Flt3)-internal tandem duplications (ITD), but not by Flt3 wild-type receptor (Flt3-Wt) in response to Flt3 ligand (FL). Here we show that Pim2 can complement Flt3-Wt signaling and induce transformation similar to Flt3-ITD in myeloid cells. Our data demonstrate that Pim2 is necessary but not sufficient for Flt3-ITD-induced transformation of 32D cells and primary bone marrow cells as assessed by colony assays. Pim2-induced clonogenic growth of FL-treated 32D-Flt3-Wt cells. Proliferation of 32D-Flt3-Wt cells was significantly enhanced in FL-treated Pim2-overexpressing cells. This increase was associated with enhanced S-phase cell cycle progression. Pim2-overexpressing cells were resistant to apoptosis induced by growth factor deprivation or treatment with tyrosine kinase inhibitor (PKC412). The Flt3 point mutant D835Y, which is not able to support colony growth of myeloid cells, also induced clonogenic growth in the presence of Pim2. In conclusion, Pim2 is an important target of Flt3-ITD-induced transformation, and overexpression of Pim2 together with Flt3-Wt or D835Y receptor mimics Flt3-ITD-mediated transformation. Pim2 complements with Flt3-Wt signaling to induce proliferation by enhancing G(1)/S-phase progression of the cell cycle.