Human endometrial mesenchymal stem cells (eMSCs) are a novel source of MSC easily harvested from endometrial biopsies. In preparation for clinical application, we have developed protocols for eMSC isolation from single cell suspensions using magnetic bead sorting with a perivascular marker, SUSD2 and culture expansion in serum free medium (SFM) (1,2). Like other MSC, eMSC undergo spontaneous differentiation into fibroblasts during culture expansion, decreasing their purity and efficacy. The aim of this study was to determine whether A83-01, a TGFb receptor inhibitor with specificity for ALK4/5/7, prevents differentiation of eMSC in culture. SUSD2+ eMSCs were cultured in SFM with bFGF and EGF in 5%O2/5%CO2 until passage 5 (P5). P6 cells were incubated with or without A83-01 for 7 days, then analysed for MSC properties. Flow cytometry was used to quantify autofluorescence, examine cell cycle status and assess apoptosis using AnnexinV. A83-01 dose dependently promoted SUSD2+ cell proliferation with a maximal effect at 1uM. A83-01 increased the %SUSD2+ cells in P6 cultures (P<0.005 n=9). A83-01-treated cells had higher cloning efficiency (p=0.03 n=9), differentiated into mesodermal lineages and expressed MSC but not pluripotency markers. More A83-01-treated P6 eMSCs were present in the G2/M and fewer in the subG0/G1 peaks of Hoechst-stained cells (both p<0.02 n=7). Fewer A83-01-treated cells expressed AnnexinV (P<0.05 n=6). Fewer A83-01 treated cells were autofluorescent (p=0.001 n=14) or stained with β-galactosidase, senescence markers. These data suggest that A83-01 promotes SUSD2+ cell proliferation and blocks senescence and apoptosis in late passage cultures. Decreased SMAD2/3 phosphorylation in A83-01-treated SUSD2+ cells indicates that A83-01 binding to TGFβ receptors blocks downstream signalling leading to apoptosis. Small molecules such as A83-01 that promote eMSC proliferation in the undifferentiated state may provide an approach for the expansion of undifferentiated MSC for use in tissue engineering and cell-based therapies for gynaecological applications.