Recently, femtosecond laser pulses have been utilized for the targeted introduction of genetic matter into mammalian cells. This rapidly expanding and developing novel optical technique using a tightly focused laser light beam is called phototransfection. Extending previous studies [Stevenson et al., Opt. Express, 7125–7133 (2006)], we show that femtosecond lasers can be used to phototransfect a range of different cell lines, and specifically that this novel technology can also transfect mouse embryonic stem cell colonies with efficiency. Notably, we show the ability of differentiating these cells into the extraembryonic endoderm using phototransfection. Furthermore, we present two new findings aimed at optimizing the phototransfection method and improving applicability: first, the influence of the cell passage number on the transfection efficiency is explored and, second, the ability to enhance the transfection efficiency via whole culture treatments. Our results should encourage wider uptake of this methodology.