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She was an illegitimate daughter of King James IV of Scotland who served as governess to her half-niece Mary, Queen of Scots. Janet was briefly a mistress of King Henry II of France, by whom she had a legitimated son: Henri d´Angoulême. Her daughter, Mary Fleming, was one of the young queen´s "Four Marys".
Janet Stewart (also referred to as Jane, Jenny, and other variants) was the fifth illegitimate child of the Stewart king James IV to reach adulthood. Her half-brothers included James Stewart, 1st Earl of Moray; Alexander Stewart, Lord Chancellor of Scotland; and James V, King of Scots, her father´s only surviving legitimate child.

Her mother—the fourth royal mistress of James IV to have children—was Agnes (secondarily known as Isabel), daughter of James Stewart, 1st Earl of Buchan (who bore the nickname "Hearty James"). Janet´s parents were distantly related by a common ancestor: Joan Beaufort, Queen of Scots.

Janet Stewart married Malcolm Fleming, 3rd Lord Fleming, after being granted a dispensation on 26 February 1524/5. They had eight children:

1)James Fleming, 4th Lord Fleming (died 1558), who married Barbara Hamilton
2)John Fleming, 5th Lord Fleming
3)Joanna Fleming, who first married John Livingston, Master of Livingston and eldest son of Alexander Livingston, 5th Lord Livingston, who was killed at the Battle of Pinkie along with her father. Sometime after 24 May 1560, she married John Sandilands of Calder, who died in May 1567, with whom she had issue. Before November 1567, she married for the third time to David Craufurd of Kerse.
4)Janet Fleming, who married Richard Brown, Younger of Hartree, who died after 23 September 1536. Their son and heir apparent was Andrew Brown of Hartree
5)Agnes Fleming (died before 18 October 1597), who married before 1 October 1553, William Livingstone, 6th Lord Livingston, and had issue
6)Margaret Fleming (died before 15 Mar 1586/7), who married Robert Graham, Lord Graham, who died at the Battle of Pinkie, and had issue, John Graham, 3rd Earl of Montrose. Sometime after 30 Jan 1548/9, she married Thomas Erskine, Master of Erskine, a son of John Erskine, 5th Lord Erskine. On 1 April 1557, she married for the third time to John Stewart, 4th Earl of Atholl.
7)Mary Fleming, a Lady-in-Waiting to Mary Queen of Scots, who married Mary´s secretary, William Maitland of Lethington. After his death, she married George Meldrum of Fyvie.
8)Elizabeth Fleming (died after 1550), who married before 24 March 1540/1 William Crichton, 5th Lord Crichton of Sanquhar, and had issue including Robert Crichton, 6th Lord Crichton of Sanquhar.