Nanog is one of key genes in the complicated gene network maintaining the undifferentiated state of embryonic stem (ES) cells. Injectoassay was applied to the evaluation of the functional activity of Nanog in viable ES single-cells. The functional sequence of Nanog was cloned from the genome DNA of a mouse ES cell and inserted into an overexpression vector together with a reporter gene of green fluorescent protein. The thus-prepared Nanog expression vector was injected into mouse ES single-cells in a semi-quantitative manner using a sub-micropipette containing a defined vector concentration. Culture was continued for 72 h in the presence or absence of the leukemia inhibitory factor (LIF) and the morphological changes of colonies were analyzed to distinguish the differentiated state from the undifferentiated state. When the Nanog expression level was sufficiently high, the undifferentiated state could be maintained for 48 h, even in the absence of LIF.