This study was designed to evaluate the in vitro chondrogenic differentiation potential of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs) in response to different culture conditions. Fat tissue cells after collection were cultured in optimized conditions until passage 3 (P3). P3 cells were cultured as micropellets under different chondrogenic conditions for 21 days in 5 groups: basic medium as a control, basic chondrogenic medium (BCM), BCM supplemented with transforming growth factor beta 3 (TGF-β3; 10 ng/mL), BCM supplemented with bone morphogenetic protein 6 (BMP-6; 10 ng/mL), and BCM supplemented with both TGF-β3 and BMP-6. The growth rate of pellets was measured and differentiation progression was assessed by specific stainings and gene expression profiling. Results showed that the largest pellets of cells belonged to the group containing both growth factors. All treated cells showed different levels of chondrogenic differentiation compared to the control group (P < 0.05). The differentiation score in groups containing TGF-β3 and both growth factors was significantly greater than that in other groups. Moreover, the expression of aggrecan, as a cartilage-specific gene, was confirmed in all groups expect the control group. Taken together, these results indicate that the addition of both TGF-β3 and BMP-6 to BCM potentiates the chondrogenic differentiation of equine AT-MSCs.