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Mutations in the c-Kit gene (KIT) that result in constitutive tyrosine kinase activity are associated with mastocytosis/mast cell leukemia, gastrointestinal stromal tumors (GIST), and acute myeloid leukemia (AML). Expression of a murine Kit allele with a mutation in the phosphotransferase domain (Kit D814V) renders murine Ba/F3 cells factor-independent and causes lymphocytic leukemia in mice. An animal model using the homologous human allele (KIT D816V) has not been described. We generated retroviral expression constructs using wild-type and mutant human KIT and murine Kit that achieved equivalent protein levels in human-derived 293T cells. When expressed in murine cells however, despite equivalent transcript levels, the KITD816V protein specifically was present in lower abundance, and unlike the murine mutant, the human D816V was unable to transform Ba/F3 cells without prior selection. In contrast, human KITD816V rapidly transformed human TF-1 cells to factor independence. Fusion of the extracellular and transmembrane domains of murine Kit to the intracellular domain of human KIT (Hybrid KIT WT and Hybrid KITD816V) facilitated higher protein expression in murine cells, and rapid transformation of both Ba/F3 and TF-1 cells. Endoglycosidase digestion revealed that both human and murine mutant Kit receptors were incompletely glycosylated, and by immunohistochemistry we found that the murine and hybrid mutant KIT receptors were predominantly localized to the golgi. However, the human KIT D816V protein, in murine cells, failed to traffic past the endoplasmic reticulum (ER), while in human cells, KITD816V was able to traffic through the ER to the golgi. In murine cells expressing human KITD816V, downstream signaling pathways, including Stat3, Stat5 and Akt were activated to a lesser extent compared to cells expressing the murine KitD814V or hybrid KITD816V. Treatment of cells with small molecule inhibitors of protein trafficking demonstrated that activation of downstream signaling pathways activated by Hybrid KITD816V required golgi but not cell membrane localization. Lastly, mice transplanted with bone marrow transduced with the human KITD816V remained free of disease while mice transplanted with the golgi-localized Hybrid KITD816V succumbed to fatal myeloproliferative disease (MPD) with 100% penetrance. Taken together, our data demonstrate that murine and human cells traffic the KITD816V protein differently and that leukemogenic signals require localization of the mutant receptor to the golgi apparatus. This is the first report of an animal model of myeloid leukemia/MPD using the KIT oncogene and will be useful for elucidating the mechanisms by which KITD816V contributes to leukemogenesis and for testing novel therapeutic strategies.