The most widely used method to alter the genome of embryonic stem (ES) cells is to introduce a specifically designed DNA fragment using electroporation. The DNA will then integrate into the genome of ES cells. Colonies of cells containing the exogenous DNA are then picked, expanded, replica-plated, frozen in 96-well plates, and used as a source for genomic DNA preparation for genotyping. After ES candidate clones are identified by genomic Southern blot or polymerase chain reaction (PCR), the method of rescue of the cells from the frozen 96-well plates is very important. This protocol describes a method for thawing such cells.