Abstract Oncogenic mutations leading to persistent kinase activities are associated with malignancies. Therefore, deciphering the signaling networks downstream of these oncogenic stimuli remains a challenge to gather insights into targeted therapy. To elucidate the biochemical networks connecting the Kit mutant to leukemogenesis, in the present study, we performed a global profiling of tyrosine-phosphorylated proteins from mutant Kit-driven murine leukemia proerythroblasts and identified Shp2 and Stat5 as proximal effectors of Kit. Shp2 or Stat5 gene depletion by sh-RNA, combined with pharmacologic inhibition of PI3kinase or Mek/Erk activities, revealed 2 distinct and independent signaling pathways contributing to malignancy. We demonstrate that cell survival is driven by the Kit/Shp2/Ras/Mek/Erk1/2 pathway, whereas the G1/S transition during the cell cycle is accelerated by both the Kit/Stat5 and Kit/PI3K/Akt pathways. The combined use of the clinically relevant drugs NVP-BEZ235, which targets the cell cycle, and Obatoclax, which targets survival, demonstrated synergistic effects to inhibit leukemia cell growth. This synergy was confirmed with a human mast leukemia cell line (HMC-1.2) that expresses mutant Kit. The results of the present study using liquid chromatography/tandem mass spectrometry analysis have elucidated signaling networks downstream of an oncogenic kinase, providing a molecular rationale for pathway-targeted therapy to treat cancer cells refractory to tyrosine kinase inhibitors.