Gene targeting by homologous recombination is a powerful tool to precisely manipulate the genome in order to study the function of a gene of interest (GOI). Indeed, it has become a routine methodology in yeasts, murine embryonic stem cells, and a chicken DT40 cell line. However, gene targeting has not been used often in human somatic cells to date since the relative efficiency of gene targeting (the ratio of homologous integrations to random integrations) is remarkably low. In this review, we introduce a fundamental strategy and a protocol to generate a null allele and/or 'tetracycline-inducible conditional gene knochout' for the GOI by gene targeting in the human Nalm-6 pre-B cell line. The Nalm-6 is a rare cell line in which gene targeting by homologous recombination takes place efficiently, and it carries a stable near-diploid karyotype with a doubling time of around 20 h. In addition, the tetracycline-regulated gene depletion (Tet-Off) system is steadily applicable to this cell line. Therefore, gene targeting systems using the Nalm-6 cell are used increasingly and offer promise in the study of human gene functions. This review should prove useful to researchers in a wide rage of fields.