Interactions of multiple genes and associated factors are involved in the differentiation and de-differentiation of embryonic stem (ES) cells. Quantitative analysis of these genes and factors is essential for the elucidation of their mechanism. To meet this requirement, we have investigated various experimental conditions for high performance microinjection into mouse ES cells. A speedy and rhythmic operation was found to be important and was accomplished robotically by using a single-cell manipulation technique and XY-address registrable culture dishes. Among many experimental parameters, the tip size of an injection capillary, the pressure condition, and the DNA concentration in the injection capillary were of critical significance. Their optimum values were 0.5–0.8 μm, 0.7 kgf/cm2 for 30 ms, and 1–100 ng/μl, respectively. Under these conditions, semi-quantitative control of the EGFP gene expression in mouse ES cells and its knockdown was successfully demonstrated.