This paper describes a highly efficient method for generating conditional knockout (cko) mutations in mice using recombineering. The method involves using the λ phage Red proteins to mediate homologous recombination, allowing subcloning of DNA from BACs into high-copy plasmids via gap repair. Cre or Flpe recombinases are then used to introduce *loxP* or *FRT* sites into the subcloned DNA. Unlike methods that use short homology regions (45-55 bp), this method uses longer homology regions (200-500 bp), which significantly increases the efficiency and reliability of the process. The authors also describe new *E. coli* strains that express the required proteins from a defective λ prophage and arabinose-inducible promoters for Cre or Flpe recombinases. Two new Neo selection cassettes are introduced, which work well in both *E. coli* and mouse embryonic stem (ES) cells. The method is fast, efficient, and reliable, making it possible to generate knock-in mutations, transgene constructs, and to analyze regulatory elements and functional domains in or near genes. The authors demonstrate the utility of this method by constructing a cko-targeting vector for the Evi9 gene, which encodes a zinc finger transcription factor mutated in mouse and human hematopoietic tumors.This paper describes a highly efficient method for generating conditional knockout (cko) mutations in mice using recombineering. The method involves using the λ phage Red proteins to mediate homologous recombination, allowing subcloning of DNA from BACs into high-copy plasmids via gap repair. Cre or Flpe recombinases are then used to introduce *loxP* or *FRT* sites into the subcloned DNA. Unlike methods that use short homology regions (45-55 bp), this method uses longer homology regions (200-500 bp), which significantly increases the efficiency and reliability of the process. The authors also describe new *E. coli* strains that express the required proteins from a defective λ prophage and arabinose-inducible promoters for Cre or Flpe recombinases. Two new Neo selection cassettes are introduced, which work well in both *E. coli* and mouse embryonic stem (ES) cells. The method is fast, efficient, and reliable, making it possible to generate knock-in mutations, transgene constructs, and to analyze regulatory elements and functional domains in or near genes. The authors demonstrate the utility of this method by constructing a cko-targeting vector for the Evi9 gene, which encodes a zinc finger transcription factor mutated in mouse and human hematopoietic tumors.