Stem cells can be isolated from various tissues, including bone marrow, dental pulp, as well as adipose tissues. Due to the non-invasive isolation procedure, the adipose-derived mesenchymal stem cells (ADSCs) are introduced as an alternative stem cell source for regenerative medicine. In addition, it has been shown that Notch signaling participates in the control of ADSCs’ behavior. However, those studies were performed in the heterogeneous population of ADSCs. In the present study, human adipose tissue derived single-cell clones were isolated using a cloning ring technique and characterized for their stem cell characteristics. In addition, the participation of Notch signaling on the differentiation toward osteogenic and adipogenic lineage of these adipose derived single-cell-clones was investigated. Eighty-five single cell clones were able to isolate from adipose tissues culture. However, only 14 clones were able to proliferate for further characterization. The results showed that all fourteen clones expressed embryonic and mesenchymal stem cell marker genes. All 14 clones were able to differentiate to both osteogenic and adipogenic lineages. Low adipogenic clones had higher Notch 2, 3, and 4, Jagged1, as well as Delta1 mRNA expression compared to high adipogenic clones. On the contrary, the Notch signaling component mRNA expression was not different between low and high osteogenic clones. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. Inhibition of Notch signaling using γ-secretase inhibitor enhanced of adipogenic differentiation. Correspondingly, the initiation of Notch signaling using JAGGED1 bound surface resulted in a decrease of intracellular lipid accumulation. These results indicate that Notch signaling inhibited the adipogenic differentiation of single-cell-clone adipose derived mesenchymal stem cells.