Knockout mice have been highly useful tools in helping to understand the functional roles of specific genes in development and diseases. However, in many cases, knockout mice are embryonic lethal, which prevents investigation into a number of important questions, or they display developmental abnormalities, including fertility defects. In contrast, conditional knockout, which is achieved by the Cre-LoxP system, can be used to delete a gene in a specific organ or tissue, or at a specific developmental stage. This technique has advantages over conventional knockout, especially when conventional knockout causes embryonic lethality or when the function of maternal transcripts in early development needs to be defined. Recently, a widely used practice has been used to specifically delete genes of interest in oocytes: Zp3-Cre or Gdf9-Cre transgenic mouse lines, in which Cre-recombinase expression is driven by oocyte-specific zona pellucida 3 (Zp3) promoter or growth differentiation factor 9 (Gdf9) promoter, are crossed with mice bearing floxed target genes. This novel in vivo approach has helped to increase the understanding of the functions of specific genes in folliculogenesis/oogenesis, oocyte maturation, fertilization, and embryogenesis. In this minireview we discuss recent advances in understanding the molecular mechanisms regulating major reproductive and developmental events as revealed by oocyte-specific conditional knockout and perspectives on this technology and related studies.