Injection of transcription activator-like effector nucleases (TALEN) mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescence visualization of phosphorylated histone 2A (γH2AX) combined with fluorescence in situ hybridization revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within 6 h of injection, an activity that continued at reduced efficiency in two-cell embryos. TALEN-Agouti mRNAs injected into zygotes of brown FvB × C57BL/6 hybrid mice generated completely black pups, confirming that mutations were induced prior to, and/or early after, cell division. Founder mice, many of which were mosaic, transmitted altered Agouti alleles to F1 pups to yield an allelic series of mutant strains. Although mutations were targeted to "spacer" sequences flanked by TALEN binding sites, larger deletions that extended beyond the TALEN-binding sequences were also detected and were similarly inherited through the germ line. Zygotic coinjection of TALEN mRNAs directed to the Agouti, miR-205, and the Arf tumor suppressor loci yielded pups containing frequent and heritable mutations of two or three genes. Simultaneous gene editing in zygotes affords an efficient approach for producing mice with compound mutant phenotypes, bypassing constraints of conventional mouse knockout technology in embryonic stem cells.