The objective of this study was to define the physiologic needs of domestic cat embryos to facilitate development of a feline-specific culture medium. In a series of factorial experiments, in vivo-matured oocytes (n = 2040) from gonadotropin-treated domestic cats were inseminated in vitro to generate embryos (n = 1464) for culture. In the initial study, concentrations of NaCl (100.0 vs. 120.0 mM), KCl (4.0 vs. 8.0 mM), KH₂PO₄ (0.25 vs. 1.0 mM), and the ratio of CaCl₂ to MgSO₄-7H₂O (1.0:2.0 mM vs. 2.0:1.0 mM) in the medium were evaluated during Days 1–6 (Day 0: oocyte recovery and in vitro fertilization [IVF]) of culture. Subsequent experiments assessed the effects of varying concentrations of carbohydrate (glucose, 1.5, 3.0, or 6.0 mM; l-lactate, 3.0, 6.0, or 12.0 mM; and pyruvate, 0.1 or 1.0 mM) and essential amino acids (EAAs; 0, 0.5, or 1.0×) in the medium during Days 1–3 and Days 3–6 of culture. Inclusion of vitamins (0 vs. 1.0×) and fetal calf serum (FCS; 0 vs. 5% [v/v]) in the medium also was evaluated during Days 3–6. Development and metabolism of IVF embryos on Day 3 or Day 6 were compared to age-matched in vivo embryos recovered from naturally mated queens. A feline-optimized culture medium (FOCM) was formulated based on these results (100.0 mM NaCl, 8.0 mM KCl, 1.0 mM KH₂PO₄, 2.0 mM CaCl₂, 1.0 mM MgSO₄, 1.5 mM glucose, 6.0 mM l-lactate, 0.1 mM pyruvate, and 0× EAAs with 25.0 mM NaHCO₃, 1.0 mM alanyl-glutamine, 0.1 mM taurine, and 1.0× nonessential amino acids) with 0.4% (w/v) BSA from Days 0–3 and 5% FCS from Days 3–6. Using this medium, ~70% of cleaved embryos developed into blastocysts with profiles of carbohydrate metabolism similar to in vivo embryos. Our results suggest that feline embryos have stage-specific responses to carbohydrates and are sensitive to EAAs but are still reliant on one or more unidentified components of FCS for optimal blastocyst development.