FMS-like tyrosine kinase 3 (Flt3/CD135) is one of the most frequently mutated genes in haematological malignancies and is of clinical importance. Mutations are commonly found in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Flt3 encodes a receptor which is expressed at the surface of myeloid progenitor cells and dendritic cells (DCs). The Flt3/Flt3 ligand (Flt3L) cytokine signalling pathway is critical for DC development. Little is known about the molecular mechanisms that regulate Flt3 trafficking and expression in DCs. To address this, we have characterised Flt3 expression in primary murine dendritic cells. Our data show that Flt3 is highly expressed by DCs, in contrast to T cells and B cells. DC activation alters the expression of Flt3, with in vivo activated DCs displaying a significant reduction in Flt3 expression. In contrast, DCs activated ex vivo display a significant increase in Flt3 surface expression. To explore the trafficking of surface Flt3 in the presence of Flt3L, we have generated Flt3L tagged with a fluorescent internalisation probe. Using this probe we have quantified the internalisation kinetics of Flt3/Flt3L by resting and activated DC subsets. Finally, we have initiated analysis of DC function using a murine mouse model of the most commonly mutated form of Flt3 (Flt3-ITD). Exploring the biology of Flt3/Flt3L and DCs is critical to understanding their role in DC function in settings of health and haematological malignancy. This will, in turn, provide insights for the exploitation of the Flt3/Flt3L cytokine pathway in the development of effective drugs.