Multidrug resistance remains a significant obstacle to successful chemotherapy. The ability to determine the possible resistance mechanisms and surmount the resistance is likely to improve chemotherapy. Nilotinib is a very effective drug in the treatment of sensitive or Imatinib resistant patients. Although very successful hematologic and cytogenetics responses have been obtained in Nilotinib-treated patients, in recent years resistance cases were observed. The main objective of the project is to understand the mechanisms underlying multidrug resistance to Nilotinib to provide new targets for the treatment of chronic myeloid leukemia (CML). In this study, continuous exposure of cells to step-wise increasing concentrations of Nilotinib resulted in the selection of cells resistant to 50 nM Nilotinib and referred to as K562/NIL-50. Expression analyses of BCR-ABL gene demonstrated BCR-ABL was upregulated in resistant cells as compared to parental sensitive cells. However, nucleotide sequence analyses of ABL kinase gene revealed that there was no mutation in Nilotinib binding region of the gene in resistant cells. There was also an increase in expression levels of MRP1 gene in resistant cells, which transports the toxic substances outside of cells. Besides, Bax, which is one of the apoptosis inducing genes, was dowregulated in resistant cells. In addition to this, in resistant cells, while GCS and SK-1 genes were overexpressed, decrease in expression levels of LASS1 gene was observed. In conclusion, we determined mechanisms involved in Nilotinib resistance in CML in vitro. Targeting this mechanisms, besides inhibition of BCR-ABL may be a good way of treatment of CML.