The multidrug-resistant (MDR) phenotype is multifactorial, and cell lines presenting multiple resistance mechanisms might be good models to understand the importance of the various pathways involved. The present work characterized a MDR chronic myeloid leukemia cell line, derived from K562 through a selective process using daunorubicin. This MDR cell line was shown to be resistant to vincristine, daunorubicin, and partially resistant to imatinib. It showed a slower duplication rate. Overexpression of ABCB1 and ABCC1 was observed at the protein and functional levels and the expression of CD95, a molecule related to cell death, was reduced in the MDR cell line. Conversely, no differences were observed related to the anti-apoptotic molecule Bcl-2 or p53 expression. The activation antigen CD69 was reduced in the MDR cell line and treatment with imatinib further decreased the expressed levels. Furthermore, secretion of IL-8 was diminished in the MDR cell line. When daunorubicin-selected cells were compared to another MDR cell line, Lucena 1, derived from the same parental line K562, and selected with vincristine, a different profile was observed in relation to most aspects studied. When both cell lines were silenced for ABCB1, differences in CD69 and CD95 were maintained, despite resistance reversal. These results reinforce the idea that cell lines selected in vitro may display multiple resistance strategies that may vary with the selective agent used as well as during different steps of the selection process.