Colony-stimulating factor 2 (CSF2) regulates early embryonic development by modifying the epigenome, reducing apoptosis, and altering ratio of cells in the trophectoderm (TE) and inner cell mass (ICM) of the blastocyst. Previously, CSF2 reduced trophoblast elongation in female embryos but increased elongation in males. Here it was tested whether sexual dimorphism in response to CSF2 can be observed as early as the blastocyst stage. Embryos were produced in vitro using X- or Y-sorted sexed semen (n = 1612 putative zygotes). On d 5 of culture, droplets were supplemented with 5 µL vehicle (control) or 10 ng/mL bovine CSF2. Blastocysts (n = 210) were collected at Day 7 and labeled with a nuclear dye (Hoescht 33342; total cells) and a TE cell marker (CDX2). Number of ICM cells was calculated by subtraction. Statistical analysis was performed using the Proc Mixed procedure of SAS; data represent least squares means ± SEM. Treatment of female embryos with CSF2 increased the proportion of zygotes (P = 0.0213) and cleaved embryos (P = 0.0252) to become a blastocyst but there were no effects in males (P > 0.10). The percent of zygotes becoming blastocysts on Day 7 was 14.7 ± 2.1 vs 21.5 ± 2.1% for control and CSF2 in females and 16.2 ± 2.0 vs 16.3 ± 2.0% in males. There was no effect of CSF2 treatment, sex, or the interaction on the total cell number or number of TE (P > 0.10). There was a tendency (P = 0.0934) for ICM number to be less in females (56.2 ± 3.1 vs 61.0 ± 2.9) and the TE:ICM ratio was greater (P = 0.0217) for females (1.64 ± 0.91) compared with males (1.45 ± 0.09). Numerically (but not significantly), CSF2 tended to decrease ICM in females (53.9 ± 3.6 vs 58.6 ± 3.7) but not in males (60.4 ± 3.5 vs 61.5 ± 3.4). There was a tendency for a CSF2 by sex interaction (P = 0.0955) for TE:ICM ratio. In females CSF2 increased ratio (1.73 ± 0.11 vs 1.55 ± 0.11), but no effect was observed in males (1.41 ± 0.10 vs 1.50 ± 0.10). In conclusion, CSF2 exerts different responses on development of female and male preimplantation embryos. Consequences of actions of CSF2 on ICM and TE cell differentiation require further investigation. Support: NIH HD080855.