Embryonic development is a complex process involving integrated pathways directing the ordered proliferation, migration, and differentiation of stem cells into a developing embryo. Murine embryonic stem (mES) lines are immortal and homogeneous, and provide a relevant model of embryogenesis via embryoid body formation. An efficient gene delivery system for mES cells will facilitate investigations into the molecular events of embryonic differentiation. Here we describe the engineering and characterization of a replication-defective Herpes simplex virus-1 (HSV-1) vector, JD|[beta]||[beta]|, as a platform gene delivery vehicle. In this vector, the immediate early (IE, |[alpha]|) genes ICP4 and ICP22, and one full genomic joint element have been deleted. IE genes ICP27 and ICP0, whose low-level expression is necessary for transgene expression, have been converted into early (E, |[beta]|) genes in order to constrain their expression to ICP4-complementing cells, effectively eliminating their expression in normal cells. JD|[beta]||[beta]| does not express toxic viral genes in non-complementing cells including mES cells. JD|[beta]||[beta]| contains a green fluorescent protein (GFP) transgene under the control of the HCMV IE promoter and transduces mES cells with high efficiency. Transduction with JD|[beta]||[beta]|, even at elevated MOI, does not alter subsequent embryoid body formation nor the expression of endogenous germ layer markers during embryoid body formation, as determined by quantitative-PCR analysis. JD|[beta]||[beta]| will be further engineered to express developmentally relevant genes individually or in combination. This vector may be a useful tool for the experimental stem cell and regenerative medicine fields due to the attributes of non-toxicity, large transgene capacity, robust transgene expression, and non-integrating genome.