In vitro fertilization (IVF) involves fertilization of mature oocytes with capacitated sperm in a tissue culture dish. This technique can generate large numbers of cleavage-stage embryos without using a significant number of single-caged stud males for mating. In addition, sperm penetration is synchronous during incubation with mature oocytes, leading to synchronous development, unlike fertilization in vivo, when natural ovulation is usually spread over time. These embryos can be used for a variety of applications including rapid strain expansion and generation of age-matched experimental cohorts as well as cryopreservation at the two-cell stage (speed cryo). Another use of IVF is to generate offspring from cryopreserved sperm and from mice that, for one reason or another, will not mate or carry litters to term, that is, strain rescue. The method described here was refined at The Jackson Laboratory and has been in use for several years. There are many published alternative approaches and/or modifications, but this method, which can be used for both fresh and cryorecovered sperm, is relatively simple and effective for a wide variety of strains, although embryo yield varies considerably among strains as a function of response to superovulation and fertilization rate. Optimal results are dependent on practice and attention to detail.