Aflatoxin B1 (AFB1) is a hepatotoxic mycotoxin that contaminates food supplies worldwide. Domestic turkeys (Meleagris gallopavo) are highly susceptible to aflatoxicosis with dietary AFB1 exposure leading to liver damage, cancer, immunosuppression and secondary infections. AFB1 contamination therefore generates major economic losses for the poultry industry. To improve resistance to AFB1, the interaction between AFB1 and the immune system in turkeys needs to be better understood. AFB1 elicits an inflammatory response, yet also suppresses innate and adaptive immunity. Characterization of gene expression changes in turkeys exposed to AFB1 can elucidate gene targets for increasing resistance to aflatoxicosis. Expression profiles can also evaluate the effects of introduced amelioratory feed additives, such as the probiotic Lactobacillus (LGG). Transcriptome analysis of the spleen was conducted to specifically investigate AFB1 impacts on an immune tissue. Spleens were collected from 3 domestic turkey poults from each of 4 dietary challenge groups (control, AFB1, LGG, and AFB1 + LGG). RNA sequencing (RNA-seq) on the Illumina GA IIx was performed for 12 multiplexed RNA libraries. A combination of 76 bp single- and paired-end reads was obtained in two runs, for a total of over 100 million sequence reads. RNA-seq datasets were de novo assembled to generate predicted transcripts. The number of reads mapping to these transcripts was counted to characterize and compare the transcriptome expression level of each treatment group and to determine the effects of AFB1 and LGG. Gene expression analysis by RNA-seq provides insight into the turkey spleen transcriptome and identifies genes impacted by aflatoxicosis.