The majority of Musashi 1 (Msi1)‑positive cells derived from mouse embryonic stem cells (mESCs) are prone to differentiate into neural epithelial‑like cells, and only a small proportion of Msi1‑positive cells differentiate into intestinal epithelial‑like cells. Whether inhibiting the phosphatidylinositol 3‑kinase (PI3K) signaling of mESCs can promote the differentiation of Msi1‑positive cells into intestinal epithelial‑like cells remains to be fully elucidated. In the present study, to inhibit PI3K signaling, mESCs were treated with LY294002. A pMsi1‑green fluorescence protein reporter plasmid was used to sort the Msi1‑positive cells from mESCs treated and untreated with LY294002 (5 µmol/l). The Msi1‑positive cells were hypodermically engrafted into the backs of non‑obese diabetic/severe combined immunodeficient mice. The presence of neural and intestinal epithelial‑like cells in the grafts was detected by reverse transcription‑quantitative polymerase chain reaction analysis and immunohistochemistry. Compared with the Msi1‑positive cells derived from mESCs without LY294002 treatment, Msi1‑positive cells derived from mESCs treated with LY294002 expressed higher levels of leucine‑rich repeat‑containing G‑protein coupled receptor, a marker of intestinal epithelial stem cells, and lower levels of Nestin, a marker of neural epithelial stem cells. The grafts from Msi1‑positive cells treated with LY294002 contained more intestinal epithelial‑like tissues and fewer neural epithelial‑like tissues, compared with those from untreated Msi1‑positive cells. LY294002 had the ability to promote the differentiation of mESCs into intestinal epithelial‑like tissues. The Msi1‑positive cells selected from the cell population derived from mESCs treated with LY294002 exhibited more characteristics of intestinal epithelial stem cells than those from the untreated group.