Mouse embryonic stem cells (mESCs) are self-renewing and capable of differentiating into any of the three germ layers. An interesting feature of mESCs is the presence of cell-to-cell heterogeneity in gene expression that may be responsible for cell fate decisions. Nanog, a key transcription factor for pluripotency, displays heterogeneous expression in mESCs, via mechanisms that are not fully understood. To understand this variability, we quantitatively analyzed Nanog transcription and found that Nanog was both infrequently transcribed, and transcribed in a pulsatile and stochastic manner. It is possible that such stochastic transcriptional activation could contribute to the heterogeneity observed in Nanog expression as "intrinsic noise.'' To discriminate the effects of both intrinsic noise from other (extrinsic) noise on the expression variability of Nanog mRNA, we performed allele-specific single-molecule RNA fluorescent in situ hybridization in a reporter cell line and found that intrinsic noise contributed to approximately 45% of the total variability in Nanog expression. Furthermore, we found that Nanog mRNA and protein levels were well correlated in individual cells. These results suggest that stochastic promoter activation significantly affects the Nanog expression variability in mESCs.