This work investigates to which extent different carbon sources are metabolized and used for lipid biosynthesis in retrovirus producer cells, with the ultimate goal of understanding its importance regarding the stability/productivity of the vectors. For that purpose, isotopically labeled substrates (U-(13)C glucose, U-(13)C galactose, U-(13)C fructose, and U-(13)C glutamine) were used in combination with (13)C nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Our results show that glucose plays the major role in lipid biosynthesis, whereas glutamine, fructose and galactose are not significantly incorporated into lipids. Moreover, a correlation between medium osmolality (imposed by the presence of sorbitol) and virus stability and productivity was verified, apparently due to an enhancement in sugar metabolism. Since low stability and short half-life constitute the major bottleneck in process development for retrovirus and other enveloped viral vectors, this work presents useful knowledge for improved process robustness for these essential gene therapy vectors.