Background: Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET enzymes are able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. Furthermore, these modified bases are stable and detectable in the genome, raising the possibility that they could have epigenetic functions in their own right. To date, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture based systems, while its in vivo profile, in particular during development, remains unknown. Results: Here we describe the first analysis of the in vivo genome-wide profile of 5fC, across a range of dissected tissues from both wild type and Tdg-deficient E11.5 mouse embryos. Changes in the formylation profile of cytosine upon depletion of TDG suggest TET/TDG-mediated active demethylation occurs preferentially at intron-exon boundaries, and reveals a major role for TDG in shaping 5fC distribution at CpG islands. Moreover, we find enhancer regions exhibit high levels of 5fC, which accumulates at tissue-specific enhancers implicating a role in embryonic development. Conclusions: The tissue-specific distribution of 5fC can be regulated by the collective contribution of TET-mediated oxidation and excision by TDG. We show that the in vivo profile of 5fC during embryonic development resembles that of embryonic stem cells, sharing key features including enrichment of 5fC in enhancer and intragenic regions. Additionally, by investigating 5fC profiles in a tissue-specific manner from mouse embryos, we identified a targeted enrichment at active enhancers involved in tissue development. 5-formylcytosine has been mapped genomewide by pull-down and sequencing in mouse hindbrain, heart, carcass and liver. Each tissue was replicated in two different mice except for hindbrain which was replicated in four different mice.