In chronic myeloid leukemia (CML), resistance to imatinib is diverse. In addition to BCR-ABL-dependent mechanisms, BCR-ABL-independent mechanisms have been proposed. Here we established and characterized novel CML cell lines, an imatinib-sensitive cell line, MYL, and an imatinib-resistant subline, MYL-R. Treatment with imatinib inhibited phosphorylation of BCR-ABL and CrkL in both MYL and MYL-R, even though imatinib-induced apoptosis was preferentially observed in MYL than MYL-R, indicating that the resistance is based on a BCR-ABL-independent mechanism. MYL-R showed elevated expressions of Lyn mRNA, Lyn protein, phosphorylated Lyn, and phosphorylated STAT5. Silencing of Lyn by short-interfering RNA (siRNA) in MYL-R, but not in MYL, induced significant growth-inhibition, increased caspase-3 activity, and induced partial recovery from imatinib-resistance. Expression of Bcl-2, previously reported to be associated with Lyn-mediated resistance, was not elevated in MYL-R. Expression of Bim, which plays an important role in imatinib-induced cell-killing, was not suppressed in MYL-R. These results imply that diverse mechanisms of resistance exist among cell types. Treatment of MYL-R cells with various reagents known to have anti-leukemic activity revealed that zoledronic acid and the farnesyl transferase inhibitor (SCH 66336) showed strong synergism with imatinib; interferon alpha, PP2, CGP76030, and FK228 (depsipeptide) showed synergism; whereas soluble TRAIL and As2O3 showed additivity or antagonism, and 17-AAG and radicicol showed antagonism. Treatment with either PP2 or zoledronic acid induced greater growth-reduction in MYL-R than MYL. Taken together, Lyn may play an important role in imatinib-resistance in MYL-R. Some novel reagents, including siRNA targeting Lyn, may have good potential to overcome this resistance.