Chronic myeloid leukemia (CML) is a neoplastic disease characterized by the BCR-ABL1 fusion gene. When treated with targeted therapy using tyrosine kinase inhibitors (TKI), patients can survive for many years in remission. However, after drug administration, resistant mutations evolve in one third of the patients, often leading to disease relapse. The aim of this work was to generate BCR-ABL1 expressing cell lines to investigate the mutant subclone evolution based on a co-culture experiment with and without selection pressure of antileukemic agents. The BCR-ABL1 mutants E255V, T315I, and E255V/T315I were cloned by the so called Sleeping Beauty-system into a vector which already carried the fluorescent proteins CFP, mKeima, tdTomato or ZSGreen and were transferred by nucleofection into the Ba/F3 cell line. After three selection steps, a co-culture experiment with the cell lines T315I-CFP and E255V/T315I-tdTomato was performed. The cell lines were treated with the TKI ponatinib and the cytostatic agent hydroxyurea by incubation for 96 hours. The in vitro results showed a significant decrease of E255V/T315I mutant cells after treatment with hydroxyurea, highlighting a possibility for combinatorial and less aggressive treatment for CML-patients.