Substitution of the FLT3 “gatekeeper” phenylalanine with leucine (F691L) conferred mild resistance to ponatinib, but substitutions at the FLT3 activation loop (AL) residue D835 conferred a high degree of resistance. Saturation mutagenesis of FLT3-ITD exclusively identified FLT3 AL mutations at positions D835, D839, and Y842.