Generalized scheme for the production of aggregation chimeras. The procedure can be divided into six parts. 1. ES cells are passaged to deplete feeders. 2. ES cells are lightly trypsinized to release small clumps. ES cell clumps are transferred to drops serving as ES reservoirs on the outer rows of the aggregation plate. 3. The aggregation plate routinely contains four rows of KSOM drops: two outer rows of three drops that serve as ES cell and embryo reservoirs and two inner rows of four drops that contain depressions required for the aggregation of embryos with ES cells. Once made, the drops are overlayed with mineral oil. Depression wells are then made in the central two rows of wells of the aggregation plate. We routinely make six depressions per drop. It is advisable that the depressions are made and the plate be equilibrated in an incubator before the addition of cells or embryos. 4. The zona pellucida of the embryos is dissolved in Acid Tyrode’s solution. It is important to keep pipetting the embryos, as they tend to become sticky and should not make prolonged contact with the plastic surface of the dish, otherwise they can be difficult to dislodge. 5. The embryos are then washed through M2 medium. Zona-free embryos are transferred to a reservoir drop on the aggregation plate. Single embryos are immediately transferred into each depression. This must be done quickly, as zona-free embryos will aggregate with each other if left in contact. If tetraploid chimeras are being set up, then half of the embryos should be left in a reservoir until the ES cell clumps have been added to the embryos in the depressions. 6. ES cell clumps are transferred from the reservoirs to the depression wells. Care must be taken that the ES cells are in physical contact with the embryos, otherwise the embryos may form blastocysts that will not have incorporated the ES cells. For tetraploid aggregations, a second embryo is then placed in the depression so that the ES cells are sandwiched between two embryos.