FLT3 is a member of the class III tyrosine kinase family, consisting of five immunoglobin-like extracellular domains, a single transmembrane domain followed by a short juxtamembrane region and an interrupted kinase domain. Constitutive activation of FLT3 through mutation occurs in about 30% of AML cases. Studies show that targeted inhibition of FLT3 by small molecule tyrosine kinase inhibitors results in clinical responses in some patients. In light of the finding of resistant mutants selected by Gleevec treatment in CML, it is likely that similar mutants will arise in FLT3. Thus, the need for development of additional FLT3 inhibitors will be important to overcome this resistance. Here, we investigate a series of compounds derived from AG1295 (a FLT3 inhibitor with an IC50 of 300–500 nM) for activity against FLT3. Of these, AGS324 exhibited the most potent activity against FLT3 ITD in transfected BaF3 cells. FLT3 ITD phosphorylation was completely inhibited by treatment with AGS324 with an IC50 of ~40 nM. Likewise, downstream STAT5 and MAP kinase phosphorylation were reduced in parallel to inhibition of FLT3 activation. AGS324 inhibited proliferation of BaF3 FLT3 ITD expressing cells with an IC50 of 60 nM. IL-3 could completely rescue BaF3 ITD cells from AGS324 mediated cytotoxicity even at 10 uM of inhibitor, indicating no interference with IL-3 signaling pathways. There was also no activity against proliferation of BaF3 parental cells. Like AG1295, it showed greatly reduced activity against the D835Y point mutant for inhibition of FLT3 phosphorylation (IC50 >5 uM) and proliferation (IC50 > 10 uM). Not all cells expressing kinase domain point mutants were resistant to inhibition by AGS324. Cells expressing the D835G mutation were inhibited in a similar fashion to BaF3 ITD cells. In addition cells expressing a FLT3 Y842E activating mutation were also inhibited by AGS324. Proliferation of MV4-11 cells, derived from an AML patient, which express a FLT3 ITD mutation, was also inhibited with a similar IC50 (~80 nM). Interestingly, SEM-K2 cells, which express amplified wild-type FLT3 were relatively resistant to inhibition of proliferation and activation of FLT3 by AGS324. Several other compounds that resulted from modifications to AG1295 showed varying degrees of potency against FLT3. Taken together, these findings indicate that AGS324 is a potent inhibitor of FLT3 ITD and some kinase domain point mutations but has reduced activity against wtFLT3. Some structural modifications of AG1295 greatly increase potency against FLT3 ITD as well as some kinase domain mutations. We present some SAR data for this group of tyrphostins, which may be useful for more specific targeting of mutant FLT3.