Introduction: In order to determine the multidrug resistance (MDR) phenotype due to P-glycoprotein expression in haematological malignancies including acute myeloblastic leukemia (AML), a well-characterized P-gp expressing cell line was required to validate and standardize flow cytometric assays and to calibrate instruments. Therefore, this resistant subline of K562 was established for the first time in Iran in order to study the MDR phenotype due to P-gp expression in some cancers. Material and Methods: A resistant subline of K562 (KDI/20) to Doxorubicin from the same parental K562 was derived by stepwise increasing the concentration of Doxorubicin up to 20 ng/ml as a gold standard. For flow cytometric assessment of P-gp expression, 4E3 anti-P-gp was used. The resistant cell line was studied by rhodamine 123 for functional assay of P-gp. MDR1 gene expression was also confirmed using RT-PCR. Results: P-glycoprotein was expressed in final concentration of 20 ng/ml of Doxorubicin on 70% of K562 cells after 120 passages. The Rhodamine 123 influx was 37%. The over-expression of MDR1 gene was observed in a 30-cycle PCR. Conclusion: P-glycoprotein is expressed in human K562 cell line (K562) by continuous exposure to anticancer drug. P-glycoprotein expression is detected by several methods including flow cytometry and RT-PCR, and the number of PCR cycles is very important.