Administration of in vitro expanded mesenchymal stromal cells (MSCs) represents a promising therapy for regenerative medicine and autoimmunity. Both mouse and human MSCs ameliorate autoimmune disease in syn-, allo- and xenogeneic settings. However, MSC preparations are heterogeneous which impairs their therapeutic efficacy and endorses variability between experiments. This heterogeneity has also been a main hurdle in translating experimental MSC data from mouse models to human patients. The objective of the present manuscript has been to further characterize murine MSCs (mMSCs) with the aim of designing more efficient and specific MSC-based therapies. We have found that mMSCs are heterogeneous for endoglin (CD105) expression and that this heterogeneity is not due to different stages of MSC differentiation. CD105 is induced on a subpopulation of mMSCs early upon in vitro culture giving rise to CD105(+) and CD105(-) MSCs. CD105(+) and CD105(-) mMSCs represent independent subpopulations that maintain their properties upon several passages. CD105 expression on CD105(+) mMSCs was affected by passage number and cell confluency while CD105(-) mMSCs remained negative. The CD105(+) and CD105(-) mMSC subpopulations had similar growth potential and expressed almost identical mMSC markers (CD29(+)CD44(+)Sca1 (+) MHC-I(+) and CD45(-)CD11b(-)CD31(-)) but varied in their differentiation and immunoregulatory properties. Interestingly, CD105(-) mMSCs were more prone to differentiate into adipocytes and osteocytes and suppressed the proliferation of CD4(+) T cells more efficiently compared to CD105(+) mMSCs. Based on these studies we propose to redefine the phenotype of mMSCs based on CD105 expression.