Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical for normal hematopoiesis. Substitution of valine for aspartic acid 816 (D816V) constitutively actives human c-Kit, and this mutation is found in patients with mastocytosis, leukemia, and germ cell tumors. Immortalized murine progenitor cells (MIHCs) transduced with wild-type c-Kit proliferate in response to SCF, whereas cells expressing D816V c-Kit (MIHC-D816V) are factor-independent and tumorigenic. However, the mechanisms mediating transformation by D816V c-Kit are unknown. The objective of this study was to identify signaling components that contribute to D816V c-Kit-mediated transformation. SCF stimulates association of p85PI3K with phosphorylated tyrosine 721 of wild-type c-Kit. Phosphatidylinositol 3 kinase (PI3K) subsequently contributes to the activation of Akt and Jnks. In contrast, these studies demonstrated that the D816V c-Kit mutant was constitutively associated with phosphorylated p85PI3K, and, downstream of PI3K, Jnk 1 and Jnk 2 were activated but Akt was not. Interestingly, Erks 1 and 2 were not constitutively activated by D816V c-Kit. Thus, D816V c-Kit maintains the activity of PI3K but not of all signaling pathways activated by wild-type c-Kit. Further, all pathways downstream of PI3K are not constitutively active in MIHC-D816V cells. Studies with a PI3K inhibitor and D816V/Y721F c-Kit, a mutant incapable of recruiting PI3K, indicate that constitutive activation of PI3K through direct recruitment by D816V c-Kit plays a role in factor-independent growth of MIHC and is critical for tumorigenicity.