An array of protein supplementation is available for use in human embryo culture. Our objective was to analyze several types of protein for their ability to support mouse embryo development and implantation competence using an in vitro embryo culture and extended outgrowth model. Research Study. In vivo matured IVF mouse zygotes were cultured in sequential, medium containing 5 mg/ml of our standard recombinant albumin (Albix; ALB), and compared to 5 mg/mL albumin from four commercially available protein supplements: Vitrolife GMM (GMM), Vitrolife human serum albumin (HSA), Vitrolife HSA supplemented with additional fatty acids and carnitine (HSA+), or Quinn’s Serum Protein Substitute (SPS). On D3.5 of culture, morulae and blastocysts were placed into fibronectin coated dishes containing ICV1 (Cell Guidance Systems) medium for outgrowth. Media was replaced with IVC2 after 72h in outgrowth and embryo attachment assessed. At 120h (D8.5), embryos were fixed and measured for outgrowth area (n=40-56 per treatment). Outgrowth embryos were also stained with F-actin, DAPI, and POUF51, and then imaged using confocal microscopy to determine outgrowth volume, total cell number, and epiblast cell number, respectively (n=15-19 per treatment). Development to blastocyst on D3.5 was significantly lower in SPS than GMM, HSA+, and HSA (44.0±3.9%, 71.1±3.7%, 62.2±3.9%, 66.9±3.8%, respectively). Similarly, ALB, GMM, HSA+, and HSA all had significantly more hatching blastocysts than SPS (29.8±3.7%, 34.9±3.9%, 35.9±3.9%, 29.9±3.7%, 15.1±2.8%, respectively). There was a trend (p=0.07) towards larger outgrowth area in HSA+ and GMM compared to SPS (0.63±0.03mm2, 0.67±0.05mm2, 0.51±0.03mm2, respectively). More (p=0.05) HSA+ embryos had epiblast cells (94.4%) than those in GMM (57.9%), HSA (52.6%), or SPS (46.7%), although the average number of epiblast cells in these embryos, including those in ALB, was not different (197.5±57.5, 257.1±94.8, 202.5±91.0, 141.1±72.5, 332.8±133.8, respectively). Likewise, total cell number (889.0±264.4, ALB; 884.2±150.7, HSA+; 556.8±155.2, HSA; 526.9±90.1, GMM; 561.2±181.7, SPS) and total outgrowth volume (6.7±1.9x106 μm3, ALB; 6.1±1.8x106 μm3, HSA+; 3.8±1.1x106 μm3, HSA; 3.8±9.0x106 μm3, GMM; 3.6±1.2x106 μm3, SPS) were not significantly different between treatments. While differences were seen in developmental outcomes, type of protein supplementation did not have a statistically significant impact on implantation competence as measured in our extended outgrowth model. However, those embryos cultured in ALB and HSA+ successfully proliferated in outgrowth culture, suggesting that these two protein supplements better support blastocyst implantation potential—a hypothesis we are currently studying. This data could have an important translational impact on the choice of protein supplementation for use in human embryo culture media.