The receptor tyrosine kinase (RTK) Flt3 is expressed in early hematopoietic progenitor cells and stimulates their growth. Due to frequent mutations in the Flt3 gene in patients with acute myeloid leukemia (AML), Flt3 is regarded as a potential therapeutic target, but the underlying mechanisms are still poorly understood. Therefore, we investigated interactions of Flt3 and some Src family tyrosine kinases (SFKs), which are expressed predominantly or exclusively in hematopoietic cells and known to be involved in signal transduction by various RTKs. Employing sets of wt and mutant Flt3 and Hck, we analyzed protein binding as well as Flt3 phosphorylation and maturation in HEK-293 cells cotransfected with expression constructs encoding both binding partners. Kinase-inactive Hck-K269R was recruited to phosphotyrosine residues located in the juxtamembrane (JM) region of activated Flt3 via its SH2 domain. Several of the JM domain tyrosines were phophorylated by Hck and other SFKs. As apparent from the distribution of mature and hypoglycosylated Flt3, SFKs interfered with Flt3 maturation in a kinase-dependent manner. Together, these findings show a complex role of SFKs in Flt3 signaling and reveal a new function of SFKs in the maturation of RTKs.