The adipose-derived stromal cells (ADSCs) are capable of adipogenic differentiation by which they can be used as the seed of adipocytes and as the model to study the mechanism of differentiation. Suppressor of cytokine signaling 3 (SOCS3), a negative regulator of leptin signaling, perhaps involves in cell survival and differentiation. However, to date, rare studies are performed on ADSCs differentiation and SOCS3 regulation. In this study, a lentiviral vector expressing a shRNA targeting SOCS3 gene (Lv-shSOCS3) is developed and tested. To observe the effect of Lv-shSOCS3 on ADSCs differentiation, we infected ADSCs with Lv-shSOCS3 or Lv-shSOCS3-NC (non-silenced control lentivirus) and examined adipocyte differentiation using morphology and the expression of key gene for adipogenic differentiation using real-time PCR. The results show that Lv-shSOCS3 can significantly suppress the expression of SOCS3 mRNA and protein in ADSCs. There are no distinguishable differences on adipogenic differentiation and the expression of peroxisome proliferators-activated receptor gamma (PPARgamma) mRNA for ADSCs between infection with Lv-shSOCS3 and Lv-shSOCS3-NC. We conclude that SOCS3 knock-down does not influence the growth and adipogenic differentiation feature of ADSCs. Our study provides a new molecular basis for understanding the differentiation mechanism of ADSCs and lays the foundation for further study of the biological functions of SOCS3.