Expression of TERT, the catalytic protein subunit of the telomerase complex, can be used to generate cell lines that expand indefinitely and retain multilineage potential. We have created immortal adipose stromal cell lines (ATSCs) by stably transducing nonhuman primate-derived ATSCs with a retroviral vector expressing TERT. Transduced cells (ATSC-TERT) had an increased level of telomerase activity and increased mean telomere length in the absence of malignant cellular transformation. Long-term culture of the ATSC-TERT cells demonstrated that the cells retain the ability to undergo differentiation along multiple lineages such as adipogenic, chondrogenic, and neurogenic. Untransduced cells demonstrated markedly reduced multilineage and self-renewal potentials after 12 passages in vitro. To determine the functional role of telomerase during osteogenesis, we examined osteogenic differentiation potential of ATSC-TERT cells in vitro. Compared with naive ATSCs, which typically begin to accumulate calcium after 3-4 weeks of induction by osteogenic differentiation medium, ATSC-TERT cells were found to accumulate significant amounts of calcium after only 1 week of culture in osteogenic induction medium. The cells have increased production of osteoblastic markers, such as AP2, osteoblast-specific factor 2, chondroitin sulfate proteoglycan 4, and the tumor necrosis factor receptor superfamily, compared with control ATSCs, indicating that telomerase expression may aid in maintaining the osteogenic stem cell pool during in vitro expansion. These results show that ectopic expression of the telomerase gene in nonhuman primate ATSCs prevents senescence-associated impairment of osteoblast functions and that telomerase therapy may be a useful strategy for bone regeneration and repair.