Guided by a docking study based on a homology model of FLT3 in an active conformation, we explored structural modifications on irreversible EGFR inhibitors. This resulted in this first report of pteridin-7(8H)-one derivatives acting as potent FLT3 inhibitors. In our model, the 4-methylpiperazinyl group in the R1 substituent promotes the enzymatic and cellular activity of the compound by forming a hydrogen-bond interaction with Asn701. Conversion of the former Michael acceptor into an amino group at the 4’-position in R2 led to a noticeable improvement in FLT3 inhibition and selectivity against FLT3 over EGFR, because of the hydrogen-bond interaction between the amino group and Asp698. Substitution of a methyl group at the 3-position in R3, which may further stabilize the hydrogen-bond interaction between the 4-methylpiperazinyl group and Asn701 and a favorable intramolecular π-π stacking interaction, led to compound 31, the most potent compound in this series. This representative compound (31) has single-digit nanomolar FLT3 inhibitory and low-dose anti-AML activity. It shows great selectivity and significant binding affinity to FLT3 mutants in kinase profiling and growth inhibition activities against leukemia cell lines harboring FLT3-ITD mutants, MV4-11 and Molm13 over other leukemia and solid tumor cell lines in profiling of in vitro tumor cell lines. We also clarified the cellular cytotoxic mechanism of representative pteridin-7(8H)-one derived FLT3 inhibitors, which is mediated through inhibition of phosphorylation of FLT3 and downstream signaling proteins, and thereby induction of G0/G1 stage cell cycle arrest and apoptosis in AML cells. In the in vivo studies, 31 showed a long half-life [3.1 h (iv) and 4.5 h (po)] and suppressed tumor growth in the MV4-11 xenograft model in dose-dependent manner via oral administration. This research provides a structurally distinct scaffold for development of selective FLT3 inhibitors in treatment of AML.