We have previously identified and purified multipotent MSC-like cells in the highly regenerative endometrial lining of the human uterus (eMSC) as CD140b+CD146+ cells. Due to ease of accessibility with minimal morbidity via biopsy, we are proposing to use eMSC in cell-based therapies; however, culture conditions compliant with Good Manufacturing Practice (GMP) have not been established for eMSC. The aim of this study was to optimise serum-free and xeno-free culture conditions for expansion of eMSC for potential clinical use. Real-time cell assessment (Xcelligence) and MTS viability assays were used to measure attachment and proliferation of freshly isolated, flow cytometry sorted CD140b+CD146+ eMSC cultured in several commercially available and in-house serum-free and xeno-free media in combination with five attachment matrices (fibronectin, collagen, gelatin, laminin and Cell Start-XF(R)). Comparisons were made with standard serum-containing medium, DMEM/F-12/10 FBS. Under all conditions examined, eMSC attachment and proliferation was greatest using a fibronectin matrix, with Lonza TP-SF(R) and our in-house DMEM/SF/FGF2/EGF serum-free xeno-product containing medium similar to serum-containing medium. Hypoxia increased eMSC proliferation in DMEM/SF/FGF2/EGF serum-free medium. Culture of eMSC for 7 days on a fibronectin matrix in DMEM/SF/FGF2/EGF serum-free media in 5 O2 maintained greater numbers of undifferentiated eMSC expressing CD140b, CD146 and W5C5 compared to culture under similar conditions in Lonza TP-SF medium. However the percentage of cells expressing typical MSC phenotypic markers, CD29, CD44, CD73, CD105, were similar for both media. EMSC showed greater expansion in 2D compared to 3D culture on fibronectin-coated microbeads using the optimized DMEM/SF/FGF2/EGF medium in 5 O2. In the optimized 2D culture conditions eMSC retained CFU activity, multipotency and MSC surface phenotype, representing the first steps in their preparation for potential clinical use.