Isaac is a new, original character, not present in the FMA manga or the previous animated productions. He was expressly created for this new anime, and he was designed by 'Hiromu Arawawa' herself (the original creator).

In the Episode 58, a character resembling Dante, the main villain from the first Fullmetal Alchemist anime series is seen in a cameo appearance.

The English voice actors for Lust and Mustang, Laura Bailey and Travis Willingham, are married in real life.

There are some characters in this anime whose backdrops were not presented in any episode, but were present in the first FMA Anime. (Which does not entirely follow the story of the original Manga.) For example the character, Yoki was introduced in FMA: Brotherhood after he had already met the Elric Brothers in the first anime. His story is briefly elaborated on when he re-encounters them here. The main reason for this was to avoid too much repetition as these characters' backgrounds were already used before in the first series.

The official name for this show in Japan is "Fullmetal Alchemist" which was renamed as "Fullmetal Alchemist: Brotherhood" in the English version. The reason for the renaming was to avoid confusing it with the first series which also shared the same title.

In the English dub, the metallic 'squink' in Alphonse's voice is created by using a metal bowl perched on top of a stand. The same technique was also used in the first dub of the 2003 series. Maxey Whitehead the new voice of Alphonse also remarked that Mike McFarland has never washed the bowl to prevent any misshaping.

After parting ways with Father, Hohenheim eventually traveled to Xing and combined Alchemy with their medicinal teachings which lead to the healing science of Alkahestry.

Romi Pak, Rie Kugimiya, Kenji Utsumi, Hidekatsu Shibata, Kazuki Yao, Shouko Tsuda and Keiji Fujiwara are the only voice actors to reprise their original roles, while Yûji Ueda returns as the voice of Jean Havoc instead of Zolf Kimblee, and Nana Mizuki returns as Lan Fan instead of Wrath (not to be confused with King Bradley, as Wrath from the first anime was a completely different character and Bradley was instead the Homunculus Pride in that series).

This is the first Fullmetal Alchemist related anime to not have Aaron Dismuke as the voice of Alphonse in the English dub. This is due to his voice changing from puberty after the dubbing of the first series and was replaced by Maxey Whitehead. However he is still part of the cast as the voice of Young Van Hohenheim in Episode 40. Making this the first time he voices a Fullmetal Alchemist character who isn't Alphonse.

In the 2003 FMA, The Elric brothers leave their hometown and burn down their house on October 3, 1910. In this version, they leave and burn their house down on October 3, 1911.

In the English dub of first FMA anime and the English-released manga, Greed's henchmen, Martel (anime only), Roa, and Dolcetto were changed to Marta, Law (or Loa in the English manga), and Dorchett. In the English version of this series however they were given their original names.

Edward greatly dislikes milk, either for its taste, the fact that it is "an opaque, white liquid secreted by a cow" or both. He does, however, greatly enjoy stew and marvels that something comprised mainly of milk can taste so good. It is often joked about in both the manga and the anime the reason he is so short is because he refuses to drink milk. Incidentally, Hiromu Arakawa grew up on a dairy farm as a child.

In the volume 2 extras of the manga, Ed is asked how tall he really was. He replied that he was 165 cm (about 5'5") tall. However, this height also included his elevator shoes and hair antenna - his true height at the time was 141 cm (about 4'8"). By Chapter 84, he is shown to be taller than Winry, implying that he has at least attained his 165 cm stature.

Brice Armstrong, Dameon Clarke, Lauren Nicole Goode, and Scott McNeil were the only voice actors to not reprise their original roles or provide any voice work for this series English dub.

In this anime, Pride is the strongest and oldest homunculus, Wrath is the only aging homunculus, Sloth is the fastest, Lust is the only female, Envy is the only one who can transform and sex isn't decided, Gluttony is the youngest and weakest, Greed is the "black sheep" of the homunculus and wild card

In the first anime, Selim Bradley was a regular human, while in the original manga and this series he was the Homunculus Pride. This is because due to the slow release of the manga chapters, this fact was not acknowledged until years after the first anime had ended.

In the first anime, a dying Hohenheim leaves his family because he does not want them to witness his death. The opposite is true in this anime, as Hohenheim is immortal and leaves his family in order to find a way for him to die.