Equal expression of X-linked genes such as G6PD and PGK in females and males and the initiation of X-chromosome inactivation are critically dependent on the expression of the X-inactive specific transcript (Xist). The objective of the present study was to determine the effects of in vitro production (IVP) and nuclear transfer (NT) on the relative abundance (RA) of the X-linked transcripts G6PD, PGK, and Xist in preimplantation bovine embryos. In experiment 1, sex-determined IVP or in vivo-produced embryos were analyzed for mRNA expression of the 3 genes. The sex ratio was 36% vs. 64% in IVP blastocysts and thus deviated significantly from the expected ratio of 50% in the vivo control group. The RA of G6PD transcripts was significantly higher in female IVP embryos than in male embryos. In contrast, no significant differences were seen between in vivo-derived female embryos and their male counterparts. At the morula stage, female IVP embryos transcribed significantly more PGK mRNA than did male embryos. However, blastocysts did not exhibit significant differences in PGK transcripts. No differences were observed for in vivo-derived embryos with regard to the RA of PGK transcripts. The RA of Xist mRNA was significantly higher in all female embryos than in their male counterparts. In experiment 2, IVP, in vivo-developed, NT-derived, and parthenogenetic embryos carrying two X chromosomes of either maternal and paternal origin or of maternal origin only (parthenogenotes) were analyzed for the RA of the 3 genes. In NT-derived morulae, the RA of G6PD transcripts was significantly increased compared with their IVP and in vivo-generated counterparts. G6PD transcript levels were significantly increased in IVP blastocysts compared with in vivo-generated and parthenogenetic embryos. At the morula stage, PGK transcripts were similar in all groups, but the RA of PGK transcripts was significantly higher in IVP blastocysts than in their in vivo-generated, parthenogenetic, and NT-derived counterparts. The RA of Xist was significantly elevated in NT-derived morulae compared with IVP, in vivo-generated, and parthenogenetic embryos. NT-derived blastocysts showed an increased Xist expression compared with that of IVP, in vivo-generated, and parthenogenetic embryos. Results of the present study show for the first time that differences in X-chromosome-linked gene transcript levels are related to a perturbed dosage compensation in female and male IVP and female NT-derived embryos. This finding warrants further studies to improve IVP systems and NT protocols to ensure the production of embryos with normal gene expression patterns.