Human adipose-derived stromal cells (hASCs) are capable of multi-lineage differentiation. Co-culture of stem cells with mature cells or tissues can drive their differentiation toward required lineages. We investigated whether direct cell-to-cell contact between hASCs and human UCs, or soluble signaling molecules, could induce the differentiation of hASCs into urothelium-like cells in vitro. hASCs were isolated from human subcutaneous adipose tissue and amplified in vitro. After labeled by green fluorescent protein, hASCs were cultured with human UCs in direct co-culture, indirect co-culture and conditioned culture, respectively. Two weeks later, expressions of specific urothelial differentiation markers were analyzed by RT-PCR and immunofluorescence. We found that hASCs in direct co-culture expressed urothelial-specific genes uroplakin Ib, uroplakin II and cytokeratin 18. However, uroplakin III gene was not detected during the experimental period. Additionally, uroplakin Ib and cytokeratin 18 were observed in differentiated hASCs by immunofluorescence. Notably, none of these markers were detected in hASCs cultured in indirect co-culture and conditioned culture. These findings suggest that direct cell-to-cell contact between hASCs and human UCs can induce the differentiation of hASCs along urothelial lineage.