Many studies have investigated optimal chondrogenic conditions, but only a few of them have addressed their effects on cell viability or the methods to enhance it. This study investigated the effect of low-intensity ultrasound (LIUS), a well-known chondrogenic inducer, on the viability of human mesenchymal stem cells (hMSCs) during chondrogenic differentiation in three-dimensional (3-D) alginate culture. The hMSCs/alginate layer was cultured in a chondrogenic defined medium and treated with transforming growth factor-beta1 (TGF-beta1) and/or LIUS for 2 weeks. Along with chondrogenic differentiation for 2 weeks, the 3-D alginate culture and TGF-beta1 treatment resulted in the decrease of cell viability, which appeared to be mediated by apoptosis. In contrast, co-treatment with LIUS clearly enhanced cell viability and inhibited apoptosis under the same conditions. The effect of LIUS on the apoptotic event was further demonstrated by changes in the expression of apoptosis/viability related genes of p53, bax, bcl-2, and PCNA. These results suggest that the LIUS treatment could be a valuable tool in cartilage tissue engineering using MSCs as it enhances cell viability and directs the chondrogenic differentiation process, its well-known activity.