The invention discloses two newly-discovered Flt3 phosphorylation sites, tyrosine 589 (Tyr589) and tyrosine 591 (Tyr591) in the intracellular domain, and provides antibodies, both polyclonal and monoclonal, that selectively bind to Flt3 when phosphorylated at these novel sites. Also provided are assays utilizing these reagents, including methods for determining the phosphorylation of Flt3 in a biological sample, selecting a patient suitable for Flt3 inhibitor therapy, profiling Flt3 activation in a test tissue, and identifying a compound that modulates phosphorylation of Flt3 in a test tissue, by using a detectable reagent, such as the disclosed antibodies, that binds to Flt3 when phosphorylated at Tyr589 or Tyr591. The sample or test tissue may be taken from a subject suspected of having cancer, such as acute myelogenous leukemia (AML).