The present study was undertaken to conduct a cellular and genetic analyses of in vitro developmental potential, cell lineage allocation and sexual dimorphisms in the pre-attachment bovine embryo. The use of a chemically defined system for the differential staining of trophectoderm and inner cell mass (ICM) revealed a similar proportion of blastomeres allocated to the ICM in murine (0.27), porcine (0.21) and bovine (0.23) blastocysts. Comparison of in vivo with in vitro derived bovine embryos revealed significant (p < 0.05) differences in the number of cells allocated to both ICM and trophectoderm. A differential staining method that was suitable for analysis with both fluorescence and confocal microscopy was also developed and permitted quantification of ICM and trophectoderm nuclei. Bovine parthenotes and embryos produced by in vitro fertilization were compared for chromosomal complement and developmental potential. A similar percentage of blastocyst formation was observed on day 8 post-insemination or activation (16.4 ± 3.3%) and (15.8 ± 1.0%), respectively. Differential staining indicated a significant reduction in the number of blastomeres allocated to both ICM and trophectoderm. In parthenotes there was also a lower proportion of cells in the ICM (p < 0.05). All parthenotes were polyploid or mixoploid. Analysis of pronuclear formation and DNA replication suggested that a polyploid chromosome complement reflects ongoing karyokinesis without cytokinesis during the first mitotic cell cycle. Using first round PCR, the X inactive-specific transcript (Xist) was initially detected in bovine embryos at the 8-cell stage and consistently throughout development to the hatched blastocyst stage. Transcripts for the zinc finger protein on the X chromosome (Zfx) were detected at the 2-cell and blastocyst stages. Moreover, Zfx and Zfy transcripts were observed at the blastocyst stage. Analysis of cleavage pattern and cell number under different culture conditions revealed a higher developmental potential in embryos with 2-4 cells by 32 h post insemination. Significant differences were observed in the number of blastomeres in male embryos cultured in B2 Medium. Further characterization of sexual dimorphisms could provide a potential strategy to develop a non-invasive method for sex selection in embryos with high developmental potential.