Breastmilk stem cells have been recently shown to have multi-lineage differentiation potential and to express embryonic stem cell genes associated with pluripotency. Variation in expression of these genes between lactating women has been reported in the literature, however the factors influencing this are not well understood. In this study, we examined the effect of the stage of lactation on stem cell gene expression in breastmilk cells. Fifty lactating women from both Australia (n=25) and the USA (n=25) were screened for expression of pluripotency genes as well as other genes associated with stem cell activity using Real-Time PCR. Gene expression was compared for lactation stage as well as with cell lines representing the resting breast. Expression levels of the pluripotency genes OCT4, SOX2, NANOG and KLF4 were variable, with higher expression found earlier in lactation. Interestingly, expression of ESRRB, an embryonic stem cell gene and reprogramming factor, was consistently higher than all other pluripotency genes, and was also higher earlier in lactation. The mammary stem cell marker CD49f was highly expressed in most breastmilk samples examined irrespective of lactation stage. In marked contrast, expression of all these stem cell genes was minimal in resting breast cells. These findings reinforce previous reports of variable stem cell gene expression in breastmilk cells between women and show an effect of the stage of lactation, with the most prominent expression of some of these stem cell genes occurring early in lactation. This suggests that early lactation is an optimal window for the maximal harvest of breastmilk stem cells for both lactation research and regenerative medicine.