c-Src and c-Abl are two closely related protein kinases that constitute important anticancer targets. Despite their high sequence identity, they show different sensitivities to the anticancer drug imatinib, which binds specifically to a particular inactive conformation in which the Asp of the conserved DFG motif points outward (DFG-out). We have analyzed the DFG conformational transition of the two kinases using massive molecular dynamics simulations, free energy calculations, and isothermal titration calorimetry. On the basis of the reconstruction of the free energy surfaces for the DFG-in to DFG-out conformational changes of c-Src and c-Abl, we propose that the different flexibility of the two kinases results in a different stability of the DFG-out conformation and might be the main determinant of imatinib selectivity.