Mesenchymal stem cells (MSCs) are used for cell-based therapies because of their immunomodulatory properties. The immunomodulatory properties of adipogenic (AD) and osteogenic (OS) differentiated adipose tissue-derived MSCs (AD-MSCs) isolated from BALB/c, C57BL/6, and DBA mice were compared. Splenocytes proliferation was suppressed in the presence of AD-MSCs conditioned media in all mice. After OS differentiation, BALB/c AD-MSCs produced higher levels of TGF-beta and IL-17 and lower levels of NO than AD-MSCs isolated from C57BL/6 and DBA mice. In addition, OS differentiated AD-MSCs isolated from DBA mice produced lower levels of IL-10 than AD-MSCs isolated from C57BL/6 and DBA mice. After in vitro AD and OD differentiation, AD-MSCs isolated from each mouse produced higher levels of NO and IDO than undifferentiated cells. Additionally, AD-MSCs isolated from C57BL/6 and DBA mice produced higher levels of NO than AD-MSCs isolated from BALB/c mice. Adipose tissue-derived MSCs thus retain their immunomodulatory properties after in vitro OS and AD differentiation in a strain-dependent manner.