Human bone marrow-derived mesenchymal stromal cells (MSCs) have been investigated as a therapy for a wide variety of disease indications, because of their powerful intrinsic properties of self-regeneration, immunomodulation and multi-potency, as well as being readily available and easy to isolate and culture. Nevertheless, MSCs therapy suffers limited efficacy because of unique challenges like unfavorable microenvironmental factors, immunocompatibility, and stability. To improve some of their abilities, many studies have employed genetic engineering to transfer key genes into MSCs. Genetic modification of MSCs, both viral and nonviral methods, can be used to overexpress therapeutic proteins that complement the inherent properties. Alternatively, messenger RNA (mRNA) transfection is an effective tool to temporarily engineer MSCs and improve their modulatory properties. One major obstacle is the transfection efficiency and viability of human MSCs. Here, we evaluated two commercial RNA transfection reagents developed by Mirus Bio: TransIT-VirusGEN®, and TransIT®-mRNA by using CleanCap® EGFP mRNA as reporter mRNA. MSCs were plated in 2D flask and grown until 80% confluent. MSCs were then transfected with mRNA encoding GFP by using TransIT-VirusGEN®, a lipid and polymer transfection formulation suitable for GMP applications and specifically designed to support Cell and Gene Therapies requiring enhanced nucleic acid delivery to cells, and TransIT®-mRNA transfection reagent, in combination with the mRNA boost reagent, specifically designed to deliver large RNA in a variety of cells. The transfection complex reaction, ratio of mRNA and transfection reagents, were altered to determine optimal conditions. Transfected MSCs were incubated overnight and then transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. TransIT-VirusGEN® was more effective in terms of transfection efficiency and cell viability compared to TransIT®-mRNA. The highest transfection efficiency was obteined when the mRNA complexes were generated with the lower amount of mRNA (1 μg/mL). GFP transfected MSCs fluoresced by 18 hours post-transfection and maintained fluorescence up to 1 week, as detected by fluorescence microscopy. By flow cytometry analysis, TransIT-VirusGEN® could transfect over 90% of human MSCs compared to the lower 75% efficiency of TransIT®-mRNA.