Aberrant activation of c-Kit is involved in a number of human diseases including cancers and leukemias. Certain receptor tyrosine kinases, such as epidermal growth factor receptor, have been shown to indirectly recruit Cbl through the adapter protein Grb2, leading to receptor ubiquitination and degradation. In order to study the role of Grb2 in c-Kit degradation, a series of mutations of the Grb2 binding sites in c-Kit were generated (Y703F, Y936F, and Y703F/Y936F). Since other signal transduction molecules are also known to bind Y703 and Y936, the more selective asparagine-to-alanine (N-to-A) mutants N705A, N938A, and N705A/N938A were generated. We could clearly demonstrate that binding of Grb2 was dependent on intact phosphorylation sites Y703 and Y936. Furthermore, we could demonstrate the presence of Cbl in a complex with Grb2 and c-Kit. Thus, Grb2 is able to indirectly recruit Cbl to c-Kit. In the N-to-A mutants, Cbl phosphorylation was strongly reduced, which correlated with reduced ubiquitination of c-Kit as well as decreased internalization and degradation of the receptor. Taken together, we have demonstrated that, in addition to its role in positive signaling via the Ras/Erk pathway, Grb2 mediates c-Kit degradation through recruitment of Cbl to c-Kit, leading to ubiquitination of c-Kit followed by internalization and degradation.