Background: Adipose-derived stem cells (ADSCs) have the potential to differentiate into various cell lineages and the source to easily obtain from patients, and therefore they are promising candidates for cell therapy. However, a drawback is their limited life span during in vitro culture. Therefore, we established mouse ADSC lines through immortalization with human telomerase reverse transcriptase (hTERT)-IRES eGFP, and their stem cell properties were evaluated in vitro. Methods and Results: ADSCs isolated from adult ICR mice were infected with retroviruses harboring the hTERT-IRES eGFP genes. CD34⁺/Sca-1⁺ and CD34^-/Sca-1⁺ mADSC lines derived from a single cell were finally selected based on their morphology, proliferation rate, GFP intensity and phenotypic characterization by immunostaining. Two mADSC lines were positive for CD29, CD44, CD106 and Sca-1 but were negative for CD14, CD31, CD45, CD71, CD90 and CD117. There were no statistical differences in the growth rates between CD34⁺/Sca-1⁺ and CD34^-/Sca-1⁺ mADSC lines when evaluated by BrdU incorporation and WST-1 assay. Two mADSC lines showed the potential differentiating into adipogenic, osteogenic, endothelial and cardiomyogenic cell lineages, but the CD34^-/Sca-1⁺ mADSC line represented significantly higher osteogenic differentiation potential compared to that of the CD34⁺/Sca-1⁺ mADSC line. Moreover, two mADSC lines showed significantly higher adipogenic or osteogenic differentiation potential compared to that of primary ADSCs. Conclusions: We established two hTERT-immortalized mADSC lines have multi-differentiation potential. The CD34^-/Sca-1⁺ mADSC line exhibited a high level of osteogenic differentiation potential than the CD34⁺/Sca-1⁺ mADSC line showing that phenotypic characteristics of stem cells may reflect their differentiation potential. The hTERT-immortalized ADSC lines are very useful for the study of stem cell in vitro as well as for cell therapy in vivo.