The regulation of stem cell differentiation is key for the achievement of tissue engineering and regenerative medicine. Differentiation is regulated by an extracellular microenvironment, including the extracellular matrix (ECM), in vivo. Thus, decellularized ECM (dECM) is applied to provide an ECM that mimics the in vivo conditions. Both tissue- or organ-derived dECMs and cultured cell-derived dECMs are applied for the regulation of stem cell differentiation. The tissue- or organ-derived dECMs possess advantages in their heterogeneity due to their basic architectures, and they might induce the site-specific differentiation of stem cells. In contrast, it is feasible to prepare cultured cell-derived dECMs at specific differentiative stages, which might induce strong stem cell differentiation. Additionally, cultured cell-derived dECMs can be used as in vitro ECM models to comprehensively investigate the roles of ECM. Finally, future perspectives on the realization of dECM in stem cell differentiation are described.